Compositions and soft tissue replacement methods

ABSTRACT

The present specification discloses compositions and methods of transplanting tissue useful for treating a soft tissue condition of an individual.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to and the benefit of U.S. ProvisionalPatent Application No. 61/375,144, filed Aug. 19, 2010, the entiredisclosure of which is incorporated herein by this specific reference.

Soft tissue replacement methods are commonly used for a wide variety ofclinical and cosmetic purposes. One use involves reconstructiveapplications that rebuild and restore a body part or structure tocorrect deformities from congenital anomalies, trauma, cancer,infections, disease, or medication side effects. The replacement tissueserves to support surrounding tissue and to maintain the normalappearance of the body. The restoration of this normal appearance has anextremely beneficial psychological effect on post-operative patients,alleviating much of the shock and depression that often followsextensive surgical procedures. Another use involves augmentationapplications that alter a body part or structure usually to improve itscosmetic or aesthetic appearance. Augmentation of the appearance alsohas beneficial psychological effects that improve self-esteem,well-being, and confidence of an individual. A third use involvesstructural applications that provides support to a body part orstructure to improve its function, thereby alleviating a symptomassociated with a disorder involving soft tissue loss. Examples of suchdisorders include, without limitation, stress urinary incontinence,fecal incontinence, vocal cord paralysis, vocal atrophy, vocalimplantation, intubation trauma, post-hemilaryngectomy defects,irradiation damage, lumbar disc repair, and plantar footpad repair.

Soft tissue replacement methods currently rely on two generalapproaches: 1) implantation of artificial or alloplastic fillers likesoft tissue implants, and injectable polymers and hydrogels; and 2)transplantation of tissue like tissue flaps and autologous tissuetransfers. A drawback to the use of artificial or alloplastic fillers itthat these inorganic materials lack any metabolic activity, do notbecome physiologically incorporated into the body, and as such,surrounding tissue and blood supply does not develop within theimplanted material. In addition, artificial or alloplastic fillers riskmigration and/or extrusion from the implant site. Furthermore, many ofthese fillers produce only temporary effects because the body rapidlyreabsorbs them. Fillers providing long-term effects frequently induce aforeign body response resulting in formation of an avascular, fibrouscapsule around the filler, which limits performance, distorts theaesthetic appearance of the surrounding area, and can cause pain to theindividual. Furthermore, long-term fillers, like soft tissue implants,do not remodel with the aging tissue resulting in a material that maynot be aesthetically acceptable as the patient's tissues undergo thenormal physiologic changes associated with aging.

Although the use of transplanted tissue avoids the problems associatedwith artificial or alloplastic fillers, drawbacks are also associatedwith these procedures. In these procedures, loss of transplanted tissuevolume over time as a result of its resorption by the body is a majorproblem. For example, transplantation of adipose tissue generallyresults in a loss of 20% to 90% of it volume one year after. This tissueloss is unpredictable and is a result of poor survival of thetransplanted tissue due to necrosis and a lack of vascular formation.With respect to adipose tissue, tissue breakdown is associated withtraumatic rupture of the cells, avascular necrosis, apoptosis of theadipocytes, inflammation secondary to apoptosis, fibrosis andcontraction of the graft, and/or delipidation of the adipocytes withsubsequent volume loss. Failed tissue grafts sometime produce stellateand irregular nodules with calcifications. As such, transplanted tissuemethods are usually performed two or three times to obtain the desiredeffect, resulting in massive time and cost.

One of the major underlying causes for tissue breakdown seen intransplanted tissue methods is the lack of a blood supply sufficient tosupport the transplanted tissue. For example, alleviation of tissueischemia is critically dependent upon formation of new blood vessels.The growth of new blood vessels and associated vasculature or repair orremodeling of existing blood vessels and associated vasculature providescollateral circulation in and around an ischemic area, improves bloodflow, and alleviates the symptoms caused by the ischemia. Thus,compositions and methods that promote new blood vessel formation withina transplanted tissue are needed as this will improve the survival rateof such tissues, thereby achieving reliable long-term survival ofgrafted tissues.

The present specification provides novel compositions and soft tissuereplacement methods using such compositions that reduce tissue volumeloss by increasing the survival rate of the transplanted tissue. Thisimproved survival rate is achieved by administering a compound thatpromotes new blood vessel formation, thereby ensuring that a bloodsupply adequate to support the transplanted tissue is established.

Thus, aspects of the present specification disclose a compositioncomprising adipose tissue and a compound having the structure of formulaI,

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ alkyl; R⁶is CO₂H, CO₂R⁷, CON(R⁷)₂, CONHCH₂CH₂OH, CON(CH₂CH₂OH)₂, CH₂OR⁷,P(O)(OR⁷)₂, or

a pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable amine salt thereof; R⁷ is H, C₁-C₆ alkyl or C₂-C₆ alkenyl; Xand Y are each independently selected from H, OH, ═O, Cl, Br, I, or CF₃;Z¹ and Z² are each independently selected from CH or N; W¹ and W² areeach independently selected from CH, CH₂, aryl or substituted aryl,heteroaryl, substituted heteroaryl; m is 0 to 6; o is 0 to 4; p is 0 or1; and V is C₁-C₆ alkyl, aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl.

Other aspects of the present specification disclose a method of treatinga soft tissue condition of an individual, the method comprising the stepof administering a composition disclosed herein to a site of the softtissue condition, wherein administration of the composition promotesformation of a blood supply sufficient to support the transplantedtissue, thereby treating the soft tissue site. Non-limiting examples ofa soft tissue condition include breast imperfection, defect, diseaseand/or disorder, such as, e.g., a breast augmentation, a breastreconstruction micromastia, thoracic hypoplasia, Poland's syndrome,defects due to implant complications like capsular contraction and/orrupture; a facial imperfection, defect, disease or disorder, such as,e.g., a facial augmentation, a facial reconstruction, Parry-Rombergsyndrome, lupus erythematosus profundus, dermal divots, sunken checks,thin lips, nasal imperfections or defects, retro-orbital imperfectionsor defects, a facial fold, line and/or wrinkle like a glabellar line, anasolabial line, a perioral line, and/or a marionette line, and/or othercontour deformities or imperfections of the face; a neck imperfection,defect, disease or disorder; a skin imperfection, defect, disease and/ordisorder; other soft tissue imperfections, defects, diseases and/ordisorders, such as, e.g., an augmentation or a reconstruction of theupper arm, lower arm, hand, shoulder, back, torso including abdomen,buttocks, upper leg, lower leg including calves, foot including plantarfat pad, eye, genitals, or other body part, region or area, or a diseaseor disorder affecting these body parts, regions or areas; urinaryincontinence, fecal incontinence, other forms of incontinence; andgastroesophageal reflux disease (GERD).

Other aspects of the present specification disclose a method of treatinga soft tissue condition of an individual, the method comprising thesteps of a) administering the adipose tissue to a site of the softtissue condition; and b) administering a composition comprising acompound as disclosed herein to the site of the soft tissue condition,wherein administration of the compound promotes formation of a bloodsupply sufficient to support the transplanted tissue, thereby treatingthe soft tissue site. Non-limiting examples of a soft tissue conditioninclude those described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-7 illustrate possible ways to prepare compounds disclosedherein.

DESCRIPTION

Insufficient blood vessel formation in a repairing or otherwisemetabolically active tissue results in inadequate delivery of oxygen,nutrients, and other substances necessary to establish essentialphysiological functions to the area and promote wound healing. Theformation of blood vessels within a tissue may occur by angiogenesisand/or vasculogenesis. As used herein, the term “angiogenesis” refers toa physiological process involving the growth of new blood vessels frompre-existing vessels and includes sprouting angiogenesis, the formationof new blood vessel by sprouting off existing ones, and splittingangiogenesis (intussusception), the formation of new blood vessel bysplitting off existing ones. As used herein, the term “vasculogenesis”refers to a physiological process involving the de novo production ofnew blood-vessels by proliferating endothelial stem cells, and as such,the formation of new blood vessels when there were no pre-existing ones.

Blood vessel formation, whether angiogenesis or vasculogenesis, requiressignals form growth factors and other proteins that direct and controlthe process, such as, e.g., fibroblast growth factors (like FGF-1 andFGF-2), vascular endothelial growth factors (like VEGF-A and VEGF-C),angiopoietins (like Ang-1 and Ang-2), platelet derived growth factor(PDGF), monocyte chemotactic protein-1 (MCP-1) (also known as chemokine(C-C motif) ligand 2 (CCL-2)), transformation growth factor betas(TGF-β1, TGF-β2, TGF-β3, and TGF-β4), vascular cell adhesion molecules(like VCAM-1), matrix metalloproteinases (like MMP-2 and MPP-9),integrins, cadherins, plasminogen activators, plasminogen activatorinhibitors, and ephrin.

Of the factors listed above, two of the more critical factors essentialto new blood vessel formation are FGF-2 and VEGF. One of the mostimportant functions of fibroblast growth factor-2 (FGF-2 or bFGF) is thepromotion of endothelial cell proliferation and the physicalorganization of endothelial cells into tube-like structures, thuspromoting angiogenesis and vasculogenesis. FGF-2 is a more potent factorin new blood vessel formation than VEGF or PDGF. As well as stimulatingblood vessel growth, FGF-2 is an important factor in wound healing. Thisfactor stimulates proliferation of fibroblasts and endothelial cellsthat give rise to new vessel formation and developing granulationtissue; both increase blood supply and fill up a wound space/cavityearly in the wound-healing process.

VEGF is another major contributor to new blood vessel formation. VEGF isa major contributor to new blood vessel formation. VEGF causes a massivesignaling cascade in endothelial cells resulting in the release of manyother factors known to be responsible in new blood vessel formation. Forexample, binding to VEGF receptor-2 (VEGFR-2) starts a tyrosine kinasesignaling cascade that stimulates the production of factors thatvariously stimulate vessel permeability (eNOS, producing NO),proliferation/survival (FGF-2), migration (ICAMs/VCAMs/MMPs) and finallydifferentiation into mature blood vessels. In addition, in vitro studiesdemonstrated that upon stimulation by VEGF and FGF-2, endothelial cellswould proliferate, migrate, and eventually form tube structuresresembling capillaries.

The compounds disclosed herein, and compositions comprising suchcompounds, upregulate both FGF-2 and VEGF (see Example 2). In addition,both incisional and ulcer wound models demonstrate that the upregulationof FGF-2 and VEGF results in an improved wound healing response (seeExample 2).

Without wishing to be bound by any particular theory, the compounds,compositions and methods disclosed herein increase the survival rate oftransplanted tissue by promoting new blood vessel formation within thetransplanted tissue. Administration of a compound, either as acomposition with the transplanted tissue or as a prior or subsequentadministration to the transplantation procedure, upregulate factorsessential to new blood vessel formation, like FGF-2 and VEGF. Thisupregulation initiates the signaling cascade in endothelial cellsresponsible for the formation of new blood vessel. This initiationresults in the formation of new blood vessels that, in turn, provideoxygen, nutrients, and other substances necessary to establish essentialphysiological functions to the transplanted tissue. This adequate bloodsupply supports the establishment, growth and survival of thetransplanted tissue. Thus, the compounds, compositions and methodsdisclosed herein ameliorate or prevent one of the major underlyingcauses for the tissue breakdown of transplanted tissue, namely the lackof a blood supply sufficient to support the transplanted tissue. Inaddition, the compounds and compositions disclosed herein are superiorto the mere addition of factors critical to new blood vessel formation,like FGF-2 and VEGF, because these small molecules are more stable andless costly then these protein-based therapies. Furthermore, formationof new blood vessels can soften scar tissue, capsular contracture byfacilitating the remolding of fibrous tissue into normal soft tissuetypical of the affected region.

Aspects of the present specification provide, in part, a compositioncomprising adipose tissue. As used herein, the term “adipose tissue” issynonymous with “fat” or “fatty tissue” and refers to a loose fibrousconnective tissue comprising fat cells (adipocytes) and multiple typesof regenerative cells. Adipose tissue may comprise brown and/or whiteadipose tissue taken from any body site, such as, e.g., subcutaneous,omental/visceral, interscapular, or mediastinal. It may be obtained fromany organism having adipose tissue, or the adipose tissue used may befrom a primary cell culture or an immortalized cell line.

Adipose tissue may be collected from the same individual who isundergoing the soft tissue replacement procedure (autograft), from adonor individual who is not the same individual as the one undergoingthe soft tissue replacement procedure (allograft), or from an animalsource (xenograft). As used herein, the term “autotransplantation”refers to the transplantation of organs, tissues, or cells from one partof the body to another part in the same individual, i.e., the donor andrecipient are the same individual. Tissue transplanted by such“autologous” procedures is referred to as an autograft orautotransplant. As used herein, the term “allotransplantation” refers tothe transplantation of organs, tissues, or cells from a donor to arecipient, where the donor and recipient are different individuals, butof the same species. Tissue transplanted by such “allologous” proceduresis referred to as an allograft or allotransplant. As used herein, theterm “xenotransplantation” refers to the transplantation of organs,tissues, or cells from a donor to a recipient, where the donor is of adifferent species as the recipient. Tissue transplanted by such“xenologous” procedures is referred to as a xenograft or xenotransplant.

Adipose tissue can be collected by any procedure that can harvestadipose tissue useful for the compositions and methods disclosed herein,including, without limitation a liposuction (lipoplasty) procedure or alipectomy procedure. Procedures useful for collecting adipose tissueshould minimize the trauma and manipulation associated with adiposetissue removed. Adipose tissue may be harvested from any suitableregion, including, without limitation, a mammary region, an abdominalregion, a thigh region, a flank region, a gluteal region, a trochanterregion, or a gonadal region. Procedures useful for collecting adiposetissue are well known to a person of ordinary skill in the art. Theselected procedures may be performed concomitantly with liposculpture.

A liposuction procedure harvests adipose tissue by aspirating the tissueusing a cannula. The cannula may be connected to a syringe for manualaspiration or to a power assisted suction device, like an aspirator,adapted to collect the adipose tissue into a vacuum bottle. Aliposuction procedure does not maintain an intact blood supply of theharvested tissue. The syringe may be a 10, 20 or 60 mL syringe fittedwith a 12 or 14 gauge cannula. Non-limiting examples of liposuctionprocedures include suction-assisted liposuction (SAL),ultrasound-assisted liposuction (UAL), power-assisted liposuction (PAL),twin-cannula (assisted) liposuction (TCAL or TCL), or externalultrasound-assisted liposuction (XUAL or EUAL), or water-assistedliposuction (WAL). In addition, the liposuction procedures listed abovecan be used with any of the following procedures that vary the amount offluid injected during the procedure, such as, e.g., dry liposuction, wetliposuction, super-wet liposuction, tumescent liposuction, orlaser-assisted liposuction. An autologous soft tissue transfer proceduretypically uses adipose tissue collected from a liposuction procedure.

Although the harvested tissue may be used directly to make the disclosedcompositions, it is more typically processed to purify and/or enrich forhealthy adipocytes and regenerative cells. For example, the harvestedadipose tissue may be separated from any debris and/or contaminants suchas, e.g., blood, serum, proteases, lipases, lipids and other oils,and/or other bodily fluids; tumescent fluid and/or other materials usedin the liposuction procedure; and/or other impurities suctioned duringthe procedure. Methods useful in separating debris and/or contaminantsfrom adipose tissue useful to make the disclosed compositions,including, without limitation, centrifugation, sedimentation,filtration, and/or absorption. In addition, or alternatively, theharvested adipose tissue may be processed by washing is a physiologicalbuffer like saline to remove any debris and/or contaminants.

A lipectomy procedure harvests adipose tissue by surgical excision froma donor site in a manner that minimizes damage to the blood supply ofthe tissue using standard surgical operative procedures. This harvestedtissue is then implanted into the region needing the soft tissuereplacement. A tissue flap or tissue graft procedure typically usesadipose tissue collected from a lipectomy procedure. A tissue flap is asection of living tissue that maintained its blood supply as the tissueis moved from one area of the body to another.

A local flap uses a piece of skin and underlying tissue that lieadjacent to the wound, including adipose tissue. The flap remainsattached at one end so that it continues to be nourished by its originalblood supply, and is repositioned over the wounded area. A regional flapuses a section of tissue that is attached by a specific blood vessel.When the flap is lifted, it needs only a very narrow attachment to theoriginal site to receive its nourishing blood supply from the tetheredartery and vein. A musculocutaneous flap, also called a muscle and skinflap, is used when the area to be covered needs more bulk and a morerobust blood supply. Musculocutaneous flaps are often used in breastreconstruction to rebuild a breast after mastectomy. As an example, thetransverse rectus abdominus myocutaneous) flap (TRAM flap) is a tissueflap procedure that uses muscle, fat and skin from an abdomen to createa new breast mound after a mastectomy. This type of flap remains“tethered” to its original blood supply. In a bone/soft tissue flap,bone, along with the overlying skin, is transferred to the wounded area,carrying its own blood supply.

Typically, a wound that is wide and difficult or impossible to closedirectly may be treated with a skin graft. A skin graft is a patch ofhealthy skin that is taken from one area of the body, called the “donorsite,” and used to cover another area where skin is missing or damaged.There are three basic types of skin grafts. A split-thickness skingraft, commonly used to treat burn wounds, uses only the layers of skinclosest to the surface. A full-thickness skin graft might be used totreat a burn wound that is deep and large, or to cover jointed areaswhere maximum skin elasticity and movement are needed. As its nameimplies, a full-thickness (all layers) section of skin from the donorsite are lifted. A composite graft is used when the wound to be coveredneeds more underlying support, as with skin cancer on the nose. Acomposite graft requires lifting all the layers of skin, adipose tissue,and sometimes the underlying cartilage from the donor site.

The amount of adipose tissue collected will typically vary fromindividual to individual and can depend on a number of factorsincluding, but not limited to, amount of adipose tissue required for thesoft tissue replacement method, aesthetic expectations, age, bodyhabitus, coagulation profile, hemodynamic stability, co-morbidities, andphysician preference. A liposuction procedure may harvests form about 1mL to about 1500 mL of adipose tissue. A lipectomy procedure typicallyharvests about 1 g to about 5,000 g.

Adipose tissue comprises multiple regenerative cells. As used herein,the term “regenerative cell” refers to any cells that cause orcontribute to complete or partial regeneration, restoration, orsubstitution of structure or function of an organ, tissue, orphysiologic unit or system to thereby provide a therapeutic, structuralor cosmetic benefit. Examples of regenerative cells include stem cells,progenitor cells, and precursor cells. As used herein, the term “stemcell” refers to a multipotent regenerative cell with the potential todifferentiate into a variety of other cell types that perform one ormore specific functions and has the ability to self-renew. Some of thestem cells disclosed herein may be pluripotent. Exemplary examples ofstem cells include, without limitation, adipose-derived stem cells(ASCs; adipose-derived stromal cells), endothelial-derived stem cells(ESCs), hemopoietic stem cells (HSCs), and mesenchyma stem cells (MSCs).As used herein, the term “progenitor cell” refers to an oligopotentregenerative cell with the potential to differentiate into more than onecell type, or an unipotent regenerative cell with the potential todifferentiate into only a single cell type, that perform(s) one or morespecific functions and has limited or no ability to self-renew.Exemplary examples of progenitor cells include, without limitation,endothelial progenitor cells, keratinocytes, monoblasts, myoblasts, andpericytes. As used herein, the term “precursor cell” refers to aunipotent regenerative cell with the potential to differentiate into onecell type that performs one or more specific functions and may retainextensive proliferative capacity that enables the cells to proliferateunder appropriate conditions. Exemplary examples of precursor cellsinclude, without limitation, adipoblast (lipoblast or preadipocytes),de-differentiated adipocytes, angioblasts, endothelial precursor cells,fibroblasts, lymphoblasts, and macrophages.

Harvested adipose tissue can be supplemented with regenerative cellssuch as, e.g., stem cells, progenitor cells, and precursor cells.Regenerative cells may promote new blood vessel formation, diminishnecrosis, and/or promote a supportive microenvironment in thetransplanted tissue, thereby improving survivability of the transplantedtissue. Regenerative cells can be obtained from a variety of sources.For example, adipose tissue is rich in regenerative cells that have theability to restore and reconstruct various soft tissue defects inresponse to local differentiation clues from the recipient site. Assuch, a portion of the collected adipose tissue may be further processedin order to purify regenerative cells that can then be added back to theremainder of the harvested adipose tissue in order to enrich thismaterial for these cells. Exemplary methods describing such cellenrichment procedures can be found in, e.g., Hedrick and Fraser, Methodsof Using Adipose Tissue-Derived Cells in Augmenting Autologous FatTransfer, U.S. Patent Publication 2005/0025755, Yoshimura, et al.,Characterization of Freshly Isolated and Cultured Cells Derived from theFatty and Fluid Portions of liposuction Aspirates, J. Cell. Physiol.208: 1011-1041 (2006); Yoshimura, et al., Cell-Assisted Lipotransfer forFacial Lipoatrophy: Effects of Clinical Use of Adipose-Derived StemCells, Dermatol. Surg. 34: 1178-1185 (2008); Yoshimura, et al.,Cell-Assisted Lipotransfer for Cosmetic Breast Augmentation: SupportiveUse of Adipose-Derived Stem/Stromal Cells, Aesth. Plast. Surg. 32: 48-55(2008); each of which is hereby incorporated by reference in itsentirety.

In addition, harvested adipose tissue can be supplemented withregenerative cells obtained from cell cultures, such as, e.g., primarycell cultures and established cell cultures. For example, a portion ofharvested adipose tissue from an individual can be cultured in a mannerto produce primary cell cultures enriched for regenerative cells.Alternatively, established cell lines derived from regenerative cellsfrom adipose tissue, or another tissue source, can be cultured,harvested, and added to adipose tissue collected form an individual.Exemplary methods describing such cell culture compositions andprocedures can be found in, e.g., Casteilla, et al., Method forCulturing Cells Derived from the Adipose Tissue and Uses Thereof, U.S.Patent Publication 2009/0246182; Chazenbalk, et al, Methods of ProducingPreadipocytes and Increasing the Proliferation of Adult AdiposeStem/Progenitor Cells, U.S. Patent Publication 2009/0317367; Kleinsekand Soto, Augmentation and Repair of Sphincter Defects with CellsIncluding Adipocytic Cells, U.S. Patent Publication 2008/0299213;Rehman, et al., Secretion of Angiogenic and Antiapoptotic Factors byHuman Adipose Stromal Cells, Circulation 109: r52-r58 (2004); Kilroy, etal., Cytokine Profile of Human Adipose-Derived Stem Cells: Expression ofAngiogenic, Hematopoietic, and Pro-Inflammatory Factors, J. Cell.Physiol. 212: 702-709 (2007); each of which is hereby incorporated byreference in its entirety.

Harvested adipose tissue may be immediately used to make thecompositions disclosed herein. Alternatively, harvested adipose tissue,whether unprocessed or processed, may be stored for used at some futuredate. Harvested tissue is typically stored using a slow freezing methodof the tissue to −20° C., with or without cryopreservatives. Storedadipose tissue can typically be stored for at least 6 months.

Aspects of the present specification provide, in part, a compositioncomprising a compound having the structure of formula I

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ alkyl; R⁶is CO₂H, CO₂R⁷, CON(R⁷)₂, CONHCH₂CH₂OH, CON(CH₂CH₂OH)₂, CH₂OR⁷,P(O)(OR⁷)₂, or

a pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable amine salt thereof; R⁷ is H, C₁-C₆ alkyl or C₂-C₆ alkenyl; Xand Y are each independently selected from H, OH, ═O, Cl, Br, I, or CF₃;Z¹ and Z² are each independently selected from CH or N; W¹ and W² areeach independently selected from CH, CH₂, aryl or substituted aryl,heteroaryl, substituted heteroaryl; m is 0 to 6; o is 0 to 4; p is 0 or1; and V is C₁-C₆ alkyl, aryl, substituted aryl, heteroaryl, orsubstituted heteroaryl.

The method of preparing the compounds represented by formula I can befound in, e.g., Donde, et el., 10,10-Dialkyl Prostanoic Acid Derivativesas Agents for Lowering Intraocular Pressure, U.S. Pat. No. 6,875,787;Donde, et el., 10,10-Dialkyl Prostanoic Acid Derivatives as Agents forLowering Intraocular Pressure, U.S. Patent Publication 2004/0235958;Donde, et al., Treatment of Inflammatory Bowel Disease, U.S. PatentPublication 2005/0164992, each of which is hereby incorporated byreference in its entirety. See also companion applications Donde, etal., Compositions and Methods for Skin Repair, U.S. Provisional PatentApplication 61/374,439; and Donde, et al., Compositions and Methods forTreating Corneal Haze, U.S. Provisional Patent Application 61/369,232;each of which is incorporated by reference in its entirety.

Unless specific definitions are provided, the nomenclatures utilized inconnection with, and the laboratory procedures and techniques ofanalytical chemistry, synthetic organic and inorganic chemistrydescribed herein are those known in the art. Standard chemical symbolsare used interchangeably with the full names represented by suchsymbols. Thus, for example, the terms “hydrogen” and “H” are understoodto have identical meaning. Standard techniques may be used for chemicalsyntheses, chemical analyses, and formulation.

Any definition herein may be used in combination with any otherdefinition to describe a composite structural group. By convention, thetrailing element of any such definition is that which attaches to theparent moiety. For example, the composite group alkylamido wouldrepresent an alkyl group attached to the parent molecule through anamido group, and the term alkoxyalkyl would represent an alkoxy groupattached to the parent molecule through an alkyl group.

When ranges of values are disclosed, and the notation “from n₁ . . . ton₂” is used, where n₁ and n₂ are the numbers, then unless otherwisespecified, this notation is intended to include the numbers themselvesand the range between them. This range may be integral or continuousbetween and including the end values. By way of example, the range “from2 to 6 carbons” is intended to include two, three, four, five, and sixcarbons, since carbons come in integer units. Compare, by way ofexample, the range “from 1 to 3 μM (micromolar),” which is intended toinclude 1 μM, 3 μM, and everything in between to any number ofsignificant figures (e.g., 1.255 μM, 2.1 μM, 2.9999 μM, etc.). When n isset at 0 in the context of “0 carbon atoms”, it is intended to indicatea bond or null.

In all of the disclosed structures, straight lines represent bonds, andwhere there is no symbol for the atoms between the bonds, theappropriate carbon-containing radical is to be inferred.

As used herein, the term “alkenyl” refers to a functional groupcomprising a straight-chain or branched-chain hydrocarbon containingfrom 2 to 20 carbon atoms and having one or more carbon-carbon doublebonds and not having any cyclic structure. An alkenyl group may beoptionally substituted as defined herein. Examples of alkenyl groupsinclude, without limitation, ethenyl, propenyl, 2-methylpropenyl,butenyl, 1,4-butadienyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl,decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl,hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, eicosenyl, and thelike.

As used herein, the term “alkyl” refers to a functional group comprisinga straight-chain or branched-chain hydrocarbon containing from 1 to 20carbon atoms linked exclusively by single bonds and not having anycyclic structure. An alkyl group may be optionally substituted asdefined herein. Examples of alkyl groups includes, without limitationmethyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl,tert-butyl, pentyl, iso-amyl, hexyl, heptyl, octyl, noyl, decyl,undecyl, dodecyl tridecyl, tetradecyl, pentadecyl, hexadecyl,heptadecyl, octadecyl, nonadecyl, eicosyl, and the like.

As used herein, the term “heteroalkyl” refers to a functional groupcomprising a straight-chain or branched-chain hydrocarbon containingfrom 1 to 20 atoms linked exclusively by single bonds, where at leastone atom in the chain is a carbon and at least one atom in the chain isO, S, N, or any combination thereof. The heteroalkyl group can be fullysaturated or contain from 1 to 3 degrees of unsaturation. The non-carbonatoms can be at any interior position of the heteroalkyl group, and upto two non-carbon atoms may be consecutive, such as, e.g., —CH₂—NH—OCH₃.In addition, the non-carbon atoms may optionally be oxidized and thenitrogen may optionally be quaternized.

As used herein, the term “alkynyl” refers to a functional groupcomprising a straight-chain or branched-chain hydrocarbon containingfrom 2 to 20 carbon atoms and having one or more carbon-carbon triplebonds and not having any cyclic structure. An alkynyl group may beoptionally substituted as defined herein. Examples of alkynyl groupsinclude, without limitation, ethynyl, propynyl, hydroxypropynyl,butynyl, butyn-1-yl, butyn-2-yl, 3-methylbutyn-1-yl, pentynyl,pentyn-1-yl, hexynyl, hexyn-2-yl, heptynyl, octynyl, nonynyl, decynyl,undecynyl, dodecynyl, tridecynyl, tetradecynyl, pentadecynyl,hexadecynyl, heptadecynyl, octadecynyl, nonadecynyl, eicosynyl, and thelike.

As used herein, the term “amide” refers to a molecule comprising acarboxamide group [—C(═O)NH₂] and having the general formula RCONR′R″,wherein R, R′, and R″ are an organic moiety or group.

As used herein, the term “amidine” refers to the functional group—C(═NH)NH₂.

As used herein, the term “amido” and “carbamoyl” refer to a functionalgroup comprising an amino group attached to the parent molecular moietythrough a carbonyl group, or vice versa. As used herein, either alone orin combination, the term “C-amido” refers to a —C(═O)—NR₂ group, whereinR is an organic moiety or group. As used herein, either alone or incombination, the term “N-amido” refers to a RC(═O)NH— group, wherein Ris an organic moiety or group.

As used herein, the term “amine” refers to a molecule comprising anamino group (—NH₂) or derivative thereof. A primary amine has thegeneral formula of RNH₂, a secondary amine has the general formula ofNHRR′, a tertiary amine has the general formula of NRR′R″, wherein R,R′, and R″ are an organic moiety or group.

As used herein, the term “amino” refers to the functional group —NH₂—.

As used herein, the term “aminoalkyl” refers to a functional groupcomprising an alkyl group attached to the parent molecular moietythrough an amino group. An alkylamino group may be a mono- ordialkylated forming group such as, e.g., N-methylamino, N-ethylamino,N,N-dimethylamino, N,N-ethylmethylamino and the like.

As used herein, the term “aryl” or “aryl hydrocarbon” refers to afunctional group comprising a substituted or unsubstituted aromatichydrocarbon with a conjugated cyclic molecular ring structure of 3 to 12carbon atoms. An aryl group can be monocyclic, bicyclic or polycyclic,and may optionally include one to three additional ring structures, suchas, e.g., a cycloalkyl, a cycloalkenyl, a heterocycloalkyl, aheterocycloalkenyl, or a heteroaryl. The term “aryl” includes, withoutlimitation, phenyl (benzenyl), thiophenyl, indolyl, naphthyl, totyl,xylyl, anthracenyl, phenanthryl, azulenyl, biphenyl, naphthalenyl,1-mMethylnaphthalenyl, acenaphthenyl, acenaphthylenyl, anthracenyl,fluorenyl, phenalenyl, phenanthrenyl, benzo[a]anthracenyl,benzo[c]phenanthrenyl, chrysenyl, fluoranthenyl, pyrenyl, tetracenyl(naphthacenyl), triphenylenyl, anthanthrenyl, benzopyrenyl,benzo[a]pyrenyl, benzo[e]fluoranthenyl, benzo[ghi]perylenyl,benzo[j]fluoranthenyl, benzo[k]fluoranthenyl, corannulenyl, coronenyl,dicoronylenyl, helicenyl, heptacenyl, hexacenyl, ovalenyl, pentacenyl,picenyl, perylenyl, and tetraphenylenyl. In aspects of this embodiment,an aryl is a 3 carbon aryl, a 4 carbon aryl, a 5 carbon aryl, a 6 carbonaryl, a 7 carbon aryl, a 8 carbon aryl, a 9 carbon aryl, a 10 carbonaryl, a 11 carbon aryl, or a 12 carbon aryl.

As used herein, the term “lower aryl” refers to a functional groupcomprising a substituted or unsubstituted aromatic hydrocarbon with aconjugated cyclic molecular ring structure of 3 to 6 carbon atoms.Examples of lower aryl groups include, without limitation, phenyl andnaphthyl. In aspects of this embodiment, a lower aryl is a 3 carbonaryl, a 4 carbon aryl, a 5 carbon aryl, or a 6 carbon aryl. In otheraspects, a lower aryl is a 5 or 6 carbon aryl.

As used herein, the term “heteroaryl” refers to a functional groupcomprising a substituted or unsubstituted aromatic hydrocarbon with aconjugated cyclic molecular ring structure of 3 to 12 atoms, where atleast one atom in the ring structure is a carbon and at least one atomin the ring structure is O, S, N, or any combination thereof. Aheteroaryl group can be monocyclic, bicyclic or polycyclic, and mayoptionally include one to three additional ring structures, such as,e.g., an aryl, a cycloalkyl, a cycloalkenyl, a heterocycloalkyl, or aheterocycloalkenyl. Examples of heteroaryl groups include, withoutlimitation, acridinyl, benzidolyl, benzimidazolyl, benzisoxazolyl,benzodioxinyl, dihydrobenzodioxinyl, benzodioxolyl, 1,3-benzodioxolyl,benzofuryl, benzoisoxazolyl, benzopyranyl, benzothiophenyl,benzo[c]thiophenyl, benzotriazolyl, benzoxadiazolyl, benzoxazolyl,benzothiadiazolyl, benzothiazolyl, benzothienyl, carbazolyl, chromonyl,cinnolinyl, dihydrocinnolinyl, coumarinyl, dibenzofuranyl,furopyridinyl, furyl, indolizinyl, indolyl, dihydroindolyl, imidazolyl,indazolyl, isobenzofuryl, isoindolyl, isoindolinyl, dihydroisoindolyl,isoquinolyl, dihydroisoquinolinyl, isoxazolyl, isothiazolyl, oxazolyl,oxadiazolyl, phenanthrolinyl, phenanthridinyl, purinyl, pyranyl,pyrazinyl, pyrazolyl, pyridyl, pyrimidinyl, pyridazinyl, pyrrolinyl,pyrrolyl, pyrrolopyridinyl, quinolyl, quinoxalinyl, quinazolinyl,tetrahydroquinolinyl, tetrazolopyridazinyl, tetrahydroisoquinolinyl,thiophenyl, thiazolyl, thiadiazolyl, thienopyridinyl, thienyl,thiophenyl, triazolyl, xanthenyl, and the like. In aspects of thisembodiment, a heteroaryl is a 3 carbon heteroaryl, a 4 carbonheteroaryl, a 5 carbon heteroaryl, a 6 carbon heteroaryl, a 7 carbonheteroaryl, a 8 carbon heteroaryl, a 9 carbon heteroaryl, a 10 carbonheteroaryl, a 11 carbon heteroaryl, or a 12 carbon heteroaryl.

As used herein, the term “lower heteroaryl” refers to a functional groupcomprising a monocyclic or bicyclic, substituted or unsubstitutedaromatic hydrocarbon with a conjugated cyclic molecular ring structureof 3 to 6 atoms, where at least one atom in the ring structure is acarbon and at least one atom in the ring structure is O, S, N, or anycombination thereof. In aspects of this embodiment, a lower heteroarylis a 3 carbon heteroaryl, a 4 carbon heteroaryl, a 5 carbon heteroaryl,or a 6 carbon heteroaryl. In other aspects, a lower heteroaryl is a 5 or6 carbon heteroaryl.

As used herein, the term “cycloalkenyl” and “cycloolefin” refers to afunctional group comprising a substituted or unsubstituted non-aromatichydrocarbon with a non-conjugated cyclic molecular ring structure of 3to 12 carbon atoms having at least one carbon-carbon double bond in thecarbon ring structure. A cycloalkenyl group can be monocyclic, bicyclicor polycyclic, and may optionally include one to three additional ringstructures, such as, e.g., an aryl, a heteroaryl, a cycloalkyl, aheterocycloalkyl, or a heterocycloalkenyl. Examples of such cycloalkenylgroups include, without limitation, cyclopropene, cyclobutene,1,3-cyclobutadiene, cyclopentene, 1,3-cyclopentadiene, cyclohexene,1,3-cyclohexadiene, 1,4-cyclohexadiene, cycloheptene.1,3-cycloheptadiene, 1,4-cycloheptadiene, and 1,5-cycloheptadiene. Inaspects of this embodiment, a cycloalkenyl is a 3 carbon cycloalkenyl, a4 carbon cycloalkenyl, a 5 carbon cycloalkenyl, a 6 carbon cycloalkenyl,a 7 carbon cycloalkenyl, a 8 carbon cycloalkenyl, a 9 carboncycloalkenyl, a 10 carbon cycloalkenyl, a 11 carbon cycloalkenyl, or a12 carbon cycloalkenyl.

As used herein, the term “lower cycloalkenyl” refers to a functionalgroup comprising a monocyclic substituted or unsubstituted non-aromatichydrocarbon with a non-conjugated cyclic molecular ring structure of 3to 6 carbon atoms having at least one carbon-carbon double bond in thecarbon ring structure. In aspects of this embodiment, a lowercycloalkenyl is a 3 carbon cycloalkenyl, a 4 carbon cycloalkenyl, a 5carbon cycloalkenyl, or a 6 carbon cycloalkenyl. In other aspects, alower cycloalkenyl is a 5 or 6 carbon cycloalkenyl.

As used herein, the term “heterocycloalkenyl” refers to a functionalgroup comprising a substituted or unsubstituted non-aromatic hydrocarbonwith a non-conjugated cyclic molecular ring structure of 3 to 12 atomshaving at least one double bond, where at least one atom in the ringstructure is a carbon and at least one atom in the ring structure is O,S, N, or any combination thereof. The heterocycloalkenyl group can beunsaturated, fully saturated or contain from 1 to 3 degrees ofunsaturation. A heterocycloalkenyl group can be monocyclic, bicyclic orpolycyclic, and may optionally include one to three additional ringstructures, such as, e.g., an aryl, a heteroaryl, a cycloalkyl, aheterocycloalkyl, or a cycloalkenyl. In aspects of this embodiment, aheterocycloalkenyl is a 3 carbon heterocycloalkenyl, a 4 carbonheterocycloalkenyl, a 5 carbon heterocycloalkenyl, a 6 carbonheterocycloalkenyl, a 7 carbon heterocycloalkenyl, a 8 carbonheterocycloalkenyl, a 9 carbon heterocycloalkenyl, a 10 carbonheterocycloalkenyl, a 11 carbon heterocycloalkenyl, or a 12 carbonheterocycloalkenyl.

As used herein, the term “lower heterocycloalkenyl” refers to afunctional group comprising a monocyclic substituted or unsubstitutednon-aromatic hydrocarbon with a non-conjugated cyclic molecular ringstructure of 3 to 6 atoms having at least one double bond, where atleast one atom in the ring structure is a carbon and at least one atomin the ring structure is O, S, N, or any combination thereof. In aspectsof this embodiment, a lower heterocycloalkenyl is a 3 carbonheterocycloalkenyl, a 4 carbon heterocycloalkenyl, a 5 carbonheterocycloalkenyl, or a 6 carbon heterocycloalkenyl. In other aspects,a lower heterocycloalkenyl is a 5 or 6 carbon heterocycloalkenyl.

As used herein, the term “cycloalkyl”, “carbocyclicalkyl”, and“carbocyclealkyl” refers to a functional group comprising a substitutedor unsubstituted non-aromatic hydrocarbon with a non-conjugated cyclicmolecular ring structure of 3 to 12 carbon atoms linked exclusively withcarbon-carbon single bonds in the carbon ring structure. A cycloalkylgroup can be monocyclic, bicyclic or polycyclic, and may optionallyinclude one to three additional ring structures, such as, e.g., an aryl,a heteroaryl, a cycloalkenyl, a heterocycloalkyl, or aheterocycloalkenyl. In aspects of this embodiment, a cycloalkyl is a 3carbon cycloalkyl, a 4 carbon cycloalkyl, a 5 carbon cycloalkyl, a 6carbon cycloalkyl, a 7 carbon cycloalkyl, a 8 carbon cycloalkyl, a 9carbon cycloalkyl, a 10 carbon cycloalkyl, a 11 carbon cycloalkyl, or a12 carbon cycloalkyl.

As used herein, the term “lower cycloalkyl” refers to a functional groupcomprising a monocyclic substituted or unsubstituted non-aromatichydrocarbon with a non-conjugated cyclic molecular ring structure of 3to 6 carbon atoms linked exclusively with carbon-carbon single bonds inthe carbon ring structure. Examples of lower cycloalkyl groups include,without limitation, cyclopropyl, cyclobutyl, cyclopentyl, andcyclohexyl. In aspects of this embodiment, a lower cycloalkyl is a 3carbon cycloalkyl, a 4 carbon cycloalkyl, a 5 carbon cycloalkyl, or a 6carbon cycloalkyl. In other aspects, a lower cycloalkyl is a 5 or 6carbon cycloalkyl.

As used herein, the term “heterocycloalkyl”, “heterocyclicalkyl”, and“heterocyclealkyl” refers to a functional group comprising a substitutedor unsubstituted non-aromatic hydrocarbon with a non-conjugated cyclicmolecular ring structure of 3 to 12 atoms linked exclusively with singlebonds in the ring structure, where at least one atom in the ringstructure is a carbon and at least one atom in the ring structure is O,S, N, or any combination thereof. The heterocycloalkyl group can beunsaturated, fully saturated or contain from 1 to 3 degrees ofunsaturation. A heterocycloalkyl group can be monocyclic, bicyclic orpolycyclic, and may optionally include one to three additional ringstructures, such as, e.g., an aryl, a heteroaryl a cycloalkyl, acycloalkenyl, or a heterocycloalkenyl. A heterocycle group may beoptionally substituted unless specifically prohibited. Examples of suchheterocycloalkyl groups include, without limitation, aziridinyl,azirinyl, diazirinyl, oxiranyl, oxirenyl, dioxiranyl, thiiranyl,thiirenyl, azetidinyl, azetyl, diazetidinyl, oxetanyl, oxetyl,dioxetanyl, dioxetenyl, thietanyl, thietyl, dithietanyl, dithietyl,pyrrolidinyl, pyrrolinyl, pyrrolyl, furanyl, dihydrofuranyl,tetrahydrofuranyl, tetrahydrothiophenyl, dihydrothiophenyl, thiophenyl,imidazolidinyl, pyrazolidinyl, imidazolyl, imidazolinyl, pyrazolyl,pyrazolinyl, oxazolidinyl, isoxazolidinyl, oxazolyl, oxazolinyl,isoxazolyl, isoxazolinyl, thiazolidinyl, isothiazolidinyl, thiazolyl,thiazolinyl, isothiazolyl, isothiazolinyl, dioxolanyl, 1,3-dioxanyl,1,4-dioxanyl, dioxolanyl, 1,3-dioxolanyl, oxathiolanyl, dithiolanyl,triazolyl, dithiazolyl, furazanyl, oxadiazolyl, thiadiazolyl,tetrazolyl, piperidinyl, tetrahydropyridinyl, pyridinyl,dihydropyridinyl, dihydro[1,3]oxazolo[4,5-b]pyridinyl, pyranyl,tetrahydropyranyl, thianyl, thiopyranyl, piperazinyl, diazinyl,morpholinyl, thiomorpholinyl, oxazinyl, thiazinyl, dithianyl, dioxanyl,dioxinyl, triazinyl, trioxanyl, tetrazinyl, azepanyl, azepinyl,oxepanyl, oxepinyl, thiepanyl, thiepinyl, diazepinyl, thiazepinyl,azocanyl, azocinyl, oxecanyl, thiocanyl, and the like. In aspects ofthis embodiment, a heterocycloalkyl is a 3 carbon heterocycloalkyl, a 4carbon heterocycloalkyl, a 5 carbon heterocycloalkyl, a 6 carbonheterocycloalkyl, a 7 carbon heterocycloalkyl, a 8 carbonheterocycloalkyl, a 9 carbon heterocycloalkyl, a 10 carbonheterocycloalkyl, a 11 carbon heterocycloalkyl, or a 12 carbonheterocycloalkyl.

As used herein, the term “lower heterocycloalkyl” refers to a functionalgroup comprising a monocyclic substituted or unsubstituted non-aromatichydrocarbon with a non-conjugated cyclic molecular ring structure of 3to 6 atoms linked exclusively with single bonds in the ring structure,where at least one atom in the ring structure is a carbon and at leastone atom in the ring structure is O, S, N, or any combination thereof.Lower heterocycloalkyls may be unsaturated. Examples of lowerheterocycloalkyls include, without limitation, pyrrolidinyl,imidazolidinyl, pyrazolidinyl, piperidinyl, piperazinyl, andmorpholinyl. In aspects of this embodiment, a lower heterocycloalkyl isa 3 carbon heterocycloalkyl, a 4 carbon heterocycloalkyl, a 5 carbonheterocycloalkyl, or a 6 carbon heterocycloalkyl. In other aspects, alower heterocycloalkyl is a 5 or 6 carbon heterocycloalkyl.

As used herein, the term “functional group” refers to a specific groupof atoms within a molecule that are responsible for the characteristicchemical reactions of those molecules.

As used herein, the term “halo” or “halogen” refers to the nonmetalelements fluorine (F), chlorine (Cl), bromine (Br), iodine (I), astatine(At) and ununseptium (Us).

As used herein, the term “hydroxy” refers to the functional grouphydroxyl (—OH).

As used herein, the term “hydroxyalkyl” refers to a functional groupcomprising an alkyl group attached to the parent molecular moietythrough a hydroxyl group, such as, e.g., hydroxymethyl or hydroxyethyl.

As used herein, the term “imidate” or “imino ether” refers to a moleculecomprising an imine (C═N—) and having the general formula RN═C(OR′)R″,wherein R, R′ and R″ are an organic moiety or group.

As used herein, the term “imide” refers to a functional group comprisingtwo carbonyl groups bound to nitrogen [—C(═O)NC(═O)—], and having thegeneral formula RCONCOR′, wherein R and R′ are an organic moiety orgroup.

As used herein, the term “imine” refers to the functional group —C═NH—.A primary ketimine has the general formula RCNHR′, a secondary ketiminehas the general formula RCNR′R″, a primary aldimine has the generalformula RCNHH, and a secondary aldimine has the general formula RCNR′H,wherein R, R″ and R″ are an organic moiety or group.

As used herein, the term “imino” refers to the functional group ═NH—.

As used herein, the term “iminohydroxy” refers to the functional group═N(OH) and its corresponding anion ═N—O—.

As used herein, the term “lower” refers to a functional group ormolecule containing from 1 to 6 carbon atoms, unless otherwisespecifically defined.

As used herein, the term “nitrile” refers to a molecule comparing acyano group (—C≡N), and having the general formula RCN, wherein R is anorganic moiety or group.

As used herein, the term “nitrite” refers to the functional group —NO₂,and having the general formula RNO₂ ⁻, wherein R is an organic moiety orgroup.

As used herein, the term “nitroso” refers to the functional group —N═O,and having the general formula RNO, wherein R is an organic moiety orgroup.

As used herein, the term “nitro” refers to the functional group —NO₂,and having the general formula RNO₂, wherein R is an organic moiety orgroup.

As used herein, the term “nitrate” refers to the functional group —NO₃⁻, and having the general formula RNO₃ ⁻, wherein R is an organic moietyor group.

As used herein, the term “oxo” refers to the functional group ═O.

As used herein, the term “oxy” or “oxa” refer to the functional group—O—.

As used herein, the term “oxyalkyl” refers to a functional groupcomprising an alkyl group attached to the parent molecular moietythrough an oxy group.

As used herein, the term “unsubstituted” refers to a functional group ormolecule that has hydrogen atoms at every position on the parent chainof a hydrocarbon (e.g., —CH₂CH₃).

As used herein, the term “substituted” refers to a functional group ormolecule that has at least one substituent replacing a hydrogen atom ata position on the parent chain of a hydrocarbon. A substituted group maybe fully substituted (e.g., —CF₂CF₃), monosubstituted (e.g., —CH₂CH₂F)or substituted at a level anywhere in between fully substituted andmonosubstituted (e.g., —CH₂CH₂F, —CHFCH₂F, —CH₂ CHF₂, —CHFCHF₂).

As used herein, the term “substituent” refers to an atom or group ofatoms substituted in place of a hydrogen atom on the parent chain of ahydrocarbon. Where substituents are recited without qualification as tosubstitution, both substituted and unsubstituted forms are encompassed.Examples of substituents include, without limitation, acetyl, acyl,acylamino, acyl halide, alkenyl, alkoxy, alkyl, alkylamino,alkylcarbonyl, alkyloxy, alkyloxo, alkylthio, alkynyl, amidine, amido,amino, aryl, arylamino, arylalkenyl, arylalkoxy, arylalkyl, arylalkynyl,arylalkanoyl, aryloxy, arylthio, azide, azo, benzo, carbamyl, carbonyl,carboxyl, carboxamide, carboxamidine, cyanate, cyano, cycloalkenyl,cycloalkyl, diene, cyclodiene, disulfanyl, enone, halide, halogen,haloalkenyl, haloalkoxy, haloalkyl, heteroalkyl, heteroaryl,heterocycloalkenyl, heterocycloalkyl, hydrazinyl, hydrogen,hydroperoxide, hydroxy, hydroxyalkyl, imide, imine, imino, iminohydroxy,isocyanato, isothiocyanato, isocyanate, isocyanide, isothiocyanate,keto, mercaptyl, nitrite, nitroso, nitro, nitrate, oxo, oxy, oxoalkyl,oxyalkyl, oxime, perhaloalkoxy, perhaloalkyl, peroxy, sulfanyl,sulfhydryl, sulfinyl, sulfonyl, sulfyl, sulfonamido, thioalkyl,thiocarbony, thiocarbamyl, thiocyanate, isothiocyanate, thiocyanato,thioketo, trihalomethanesulfonamido, trihalomethoxy, and/or all lowerforms therein.

As used herein, the term “optionally substituted” refers to a functionalgroup or molecule that may be either substituted or unsubstituted.Different sets of optional substituents to a particular moiety may bedefined as needed; in these cases, the optional substitution will be asdefined, often immediately following the phrase, “optionally substitutedwith.” An optionally substituted group may be unsubstituted (e.g.,—CH₂CH₃), fully substituted (e.g., —CF₂CF₃), monosubstituted (e.g.,—CH₂CH₂F) or substituted at a level anywhere in between fullysubstituted and monosubstituted (e.g., —CH₂CH₂F, —CHFCH₂F, —CH₂ CHF₂,—CHFCHF₂).

In another embodiment, the compound has the structure of formula II,

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ alkyl; R⁴is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, a pharmaceutically acceptable saltthereof, or a pharmaceutically acceptable amine salt thereof; X and Yare each independently selected from H, OH, ═O, Cl, Br, I, or CF₃; Z¹and Z² are each independently selected from CH or N; W¹ and W² are eachindependently selected from CH, CH₂, aryl or substituted aryl,heteroaryl, substituted heteroaryl; m is 0 to 4; o is 0 to 4; p is 0 or1; and V is CH₃, aryl, aryl or substituted aryl, heteroaryl, substitutedheteroaryl.

In an aspect of this embodiment, V is

wherein U is C, N, O, or S; R⁵ is halogen, C₁-C₆ alkyl, or C₂-C₆alkenyl; and n is 0-7; and. In another aspect of this embodiment, U isS; R⁵ is F, Cl, Br, or I; and n is 1, 2, or 3. In yet another aspect ofthis embodiment W² is thiophene.

In another embodiment, the compound has the structure of formula III,

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ alkyl; R⁴is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, a pharmaceutically acceptable saltthereof, or a pharmaceutically acceptable amine salt thereof; W¹ and W²are each independently selected from CH, CH₂, aryl or substituted aryl,heteroaryl, substituted heteroaryl; m is 0 to 4; o is 0 to 4; p is 0 or1; and V is CH₃, aryl, aryl or substituted aryl, heteroaryl, substitutedheteroaryl.

In yet another embodiment, the compound has the structure of formula IV,

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ linearalkyl; R⁴ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, a pharmaceuticallyacceptable salt thereof, or a pharmaceutically acceptable amine saltthereof; m is 0 to 4; o is 0 to 4; p is 0 or 1; and V is CH₃, aryl, arylor substituted aryl, heteroaryl, substituted heteroaryl.

In still another embodiment, the compound has the structure of formulaV,

wherein each dashed line represents the presence or absence of a bond;R⁴ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl, a pharmaceutically acceptable saltthereof, or a pharmaceutically acceptable amine salt thereof; o is 0 to4; and V is CH₃, aryl, aryl or substituted aryl, heteroaryl, substitutedheteroaryl.

In a further embodiment, the compound has a structure of one of thefollowing compounds listed in Table 1, a pharmaceutically acceptablesalt thereof, or a pharmaceutically acceptable amine salt thereof.

TABLE 1 Low R_(f) High R_(f) Structure diastereomer diastereomer

34 35

36 37

38 39

40 41

42

43

44

45

46 47

48 49

50 51

52 53

54 55

56 57

58 59

60 61

62 63

64 65

66 67

68 69

70 71

72 73

74 75

In a further embodiment, the compound has a structure of compound 1, 2,3, 4, or 5, a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable amine salt thereof.

In a yet further embodiment, the compound has a structure of compound 6,a pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable amine salt thereof.

The compositions disclosed herein may, or may not, comprise any numberand combination of compounds disclosed herein. For instance, acomposition can comprise, e.g., two or more compounds, three or morecompounds, four or more compounds or five or more compounds.

The addition of a compound to an adipose tissue may be accomplished byany method that ensures sufficient distribution of the compoundthroughout the adipose tissue so as to promote formation of a bloodsupply sufficient to support the transplanted tissue. For example,mixing may occur through automated means, such as, e.g.,device-controlled agitation or centrifugation, or through manualmethods, such as, e.g., luer-locked syringes or vortexing.

The compositions disclosed herein may, or may not, comprise one or moreimmunosuppressive agents that reduce, and preferably prevent, rejectionof the transplanted tissue. As used herein, the term “immunosuppressiveagent” is synonymous with “immunosuppressive drug” and refers to acompound that inhibits or interferes with normal immune function.Exemplary immunosuppressive agents suitable with the compositions andmethods disclosed herein include, without limitation, agents thatinhibit T-cell/B-cell costimulation pathways like agents that interferewith the coupling of T-cells and B-cells via the CTLA4 and B7 pathways,including cyclosporine A, mycophenolate mofetil, rapamycin, andanti-thymocyte globulin. An immunosuppressive drug is administered in aformulation that is compatible with the route of administration and isadministered to an individual at a dosage sufficient to achieve thedesired therapeutic effect.

A compound disclosed herein, or a composition comprising such a compoundis generally administered to an individual as a pharmaceuticalcomposition. Pharmaceutical compositions may be prepared by combining atherapeutically effective amount of at least one compound according tothe present specification, or a pharmaceutically acceptable acidaddition salt thereof, as an active ingredient, with conventionalacceptable pharmaceutical excipients, and by preparation of unit dosageforms suitable for topical ocular use. The therapeutically efficientamount typically is between about 0.0001% (w/v) and about 5% (w/v),preferably about 0.001% (w/v) to about 1.0% (w/v) in liquidformulations. As used herein, the term “pharmaceutical composition” andrefers to a therapeutically effective concentration of an activecompound, such as, e.g., any of the compounds disclosed herein.Preferably, the pharmaceutical composition does not produce an adverse,allergic, or other untoward or unwanted reaction when administered to anindividual. A pharmaceutical composition disclosed herein is useful formedical and veterinary applications. A pharmaceutical composition may beadministered to an individual alone, or in combination with othersupplementary active compounds, agents, drugs or hormones. Thepharmaceutical compositions may be manufactured using any of a varietyof processes, including, without limitation, conventional mixing,dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping, and lyophilizing. The pharmaceuticalcomposition can take any of a variety of forms including, withoutlimitation, a sterile solution, suspension, emulsion, lyophilizate,tablet, pill, pellet, capsule, powder, syrup, elixir, or any otherdosage form suitable for administration.

The compounds disclosed herein may also be incorporated into a drugdelivery platform in order to achieve a controlled compound releaseprofile over time. Such a drug delivery platform comprises a compounddisclosed herein dispersed within a polymer matrix, typically abiodegradable, bioerodible, and/or bioresorbable polymer matrix. As usedherein, the term “polymer” refers to synthetic homo- or copolymers,naturally occurring homo- or copolymers, as well as syntheticmodifications or derivatives thereof having a linear, branched or starstructure. Copolymers can be arranged in any form, such as, e.g.,random, block, segmented, tapered blocks, graft, or triblock. Polymersare generally condensation polymers. Polymers can be further modified toenhance their mechanical or degradation properties by introducingcross-linking agents or changing the hydrophobicity of the sideresidues. If crosslinked, polymers are usually less than 5% crosslinked,usually less than 1% crosslinked.

Suitable polymers include, without limitation, alginates, aliphaticpolyesters, polyalkylene oxalates, polyamides, polyamidoesters,polyanhydrides, polycarbonates, polyesters, polyethylene glycol,polyhydroxyaliphatic carboxylic acids, polyorthoesters, polyoxaesters,polypeptides, polyphosphazenes, polysaccharides, and polyurethanes. Thepolymer usually comprises at least about 10% (w/w), at least about 20%(w/w), at least about 30% (w/w), at least about 40% (w/w), at leastabout 50% (w/w), at least about 60% (w/w), at least about 70% (w/w), atleast about 80% (w/w), or at least about 90% (w/w) of the drug deliveryplatform. Examples of biodegradable, bioerodible, and/or bioresorbablepolymers and methods useful to make a drug delivery platform aredescribed in, e.g., Drost, et. al., Controlled Release Formulation, U.S.Pat. No. 4,756,911; Smith, et. al., Sustained Release Drug DeliveryDevices, U.S. Pat. No. 5,378,475; Wong and Kochinke, Formulation forControlled Release of Drugs by Combining Hydrophilic and HydrophobicAgents, U.S. Pat. No. 7,048,946; Hughes, et. Al., Compositions andMethods for Localized Therapy of the Eye, U.S. Patent Publication2005/0181017; Hughes, Hypotensive Lipid-Containing BiodegradableIntraocular Implants and Related Methods, U.S. Patent Publication2005/0244464; Altman, et al., Silk Fibroin Hydrogels and Uses Thereof,U.S. patent application Ser. No. 12/764,039, filed on Apr. 20, 2010;each of which is incorporated by reference in its entirety.

In aspects of this embodiment, a polymer composing the matrix is apolypeptide such as, e.g., silk fibroin, keratin, or collagen. In otheraspects of this embodiment, a polymer composing the matrix is apolysaccharide such as, e.g., cellulose, agarose, elastin, chitosan,chitin, or a glycosaminoglycan like chondroitin sulfate, dermatansulfate, keratan sulfate, or hyaluronic acid. In yet other aspects ofthis embodiment, a polymer composing the matrix is a polyester such as,e.g., D-lactic acid, L-lactic acid, racemic lactic acid, glycolic acid,caprolactone, and combinations thereof.

One of ordinary skill in the art appreciates that the selection of asuitable polymer for forming a suitable disclosed drug delivery platformdepends on several factors. The more relevant factors in the selectionof the appropriate polymer(s), include, without limitation,compatibility of polymer with drug, desired release kinetics of drug,desired biodegradation kinetics of platform at implantation site,desired bioerodible kinetics of platform at implantation site, desiredbioresorbable kinetics of platform at implantation site, in vivomechanical performance of platform, processing temperatures,biocompatibility of platform, and patient tolerance. Other relevantfactors that, to some extent, dictate the in vitro and in vivo behaviorof the polymer include the chemical composition, spatial distribution ofthe constituents, the molecular weight of the polymer and the degree ofcrystallinity.

A drug delivery platform includes both a sustained release drug deliveryplatform and an extended release drug delivery platform. As used herein,the term “sustained release” refers to the release of a compounddisclosed herein over a period of about seven days or more. As usedherein, the term “extended release” refers to the release of a compounddisclosed herein over a period of time of less than about seven days.

In aspects of this embodiment, a sustained release drug deliveryplatform releases a compound disclosed herein with substantially firstorder release kinetics over a period of, e.g., about 7 days afteradministration, about 15 days after administration, about 30 days afteradministration, about 45 days after administration, about 60 days afteradministration, about 75 days after administration, or about 90 daysafter administration. In other aspects of this embodiment, a sustainedrelease drug delivery platform releases a compound disclosed herein withsubstantially first order release kinetics over a period of, e.g., atleast 7 days after administration, at least 15 days afteradministration, at least 30 days after administration, at least 45 daysafter administration, at least 60 days after administration, at least 75days after administration, or at least 90 days after administration.

In aspects of this embodiment, a drug delivery platform releases acompound disclosed herein with substantially first order releasekinetics over a period of, e.g., about 1 day after administration, about2 days after administration, about 3 days after administration, about 4days after administration, about 5 days after administration, or about 6days after administration. In other aspects of this embodiment, a drugdelivery platform releases a compound disclosed herein withsubstantially first order release kinetics over a period of, e.g., atmost 1 day after administration, at most 2 days after administration, atmost 3 days after administration, at most 4 days after administration,at most 5 days after administration, or at most 6 days afteradministration.

A pharmaceutical composition disclosed herein can optionally include apharmaceutically acceptable carrier that facilitates processing of anactive compound into pharmaceutically acceptable compositions. As usedherein, the term “pharmacologically acceptable carrier” is synonymouswith “pharmacological carrier” and refers to any carrier that hassubstantially no long term or permanent detrimental effect whenadministered and encompasses terms such as “pharmacologically acceptablevehicle, stabilizer, diluent, additive, auxiliary, or excipient.” Such acarrier generally is mixed with an active compound or permitted todilute or enclose the active compound and can be a solid, semi-solid, orliquid agent. It is understood that the active compounds can be solubleor can be delivered as a suspension in the desired carrier or diluent.Any of a variety of pharmaceutically acceptable carriers can be usedincluding, without limitation, aqueous media such as, e.g., water,saline, glycine, hyaluronic acid and the like; solid carriers such as,e.g., starch, magnesium stearate, mannitol, sodium saccharin, talcum,cellulose, glucose, sucrose, lactose, trehalose, magnesium carbonate,and the like; solvents; dispersion media; coatings; antibacterial andantifungal agents; isotonic and absorption delaying agents; or any otherinactive ingredient. Selection of a pharmacologically acceptable carriercan depend on the mode of administration. Except insofar as anypharmacologically acceptable carrier is incompatible with the activecompound, its use in pharmaceutically acceptable compositions iscontemplated. Non-limiting examples of specific uses of suchpharmaceutical carriers can be found in Pharmaceutical Dosage Forms andDrug Delivery Systems (Howard C. Ansel et al., eds., Lippincott Williams& Wilkins Publishers, 7^(th) ed. 1999); Remington: The Science andPractice of Pharmacy (Alfonso R. Gennaro ed., Lippincott, Williams &Wilkins, 20^(th) ed. 2000); Goodman & Gilman's The Pharmacological Basisof Therapeutics (Joel G. Hardman et al., eds., McGraw-Hill Professional,10^(th) ed. 2001); and Handbook of Pharmaceutical Excipients (Raymond C.Rowe et al., APhA Publications, 4^(th) edition 2003). These protocolsare routine and any modifications are well within the scope of oneskilled in the art and from the teaching herein.

A pharmaceutical composition disclosed herein can optionally include,without limitation, other pharmaceutically acceptable components (orpharmaceutical components), including, without limitation, buffers,preservatives, tonicity adjusters, salts, antioxidants, osmolalityadjusting agents, physiological substances, pharmacological substances,bulking agents, emulsifying agents, wetting agents, sweetening orflavoring agents, and the like. Various buffers and means for adjustingpH can be used to prepare a pharmaceutical composition disclosed herein,provided that the resulting preparation is pharmaceutically acceptable.Such buffers include, without limitation, acetate buffers, boratebuffers, citrate buffers, phosphate buffers, neutral buffered saline,and phosphate buffered saline. It is understood that acids or bases canbe used to adjust the pH of a composition as needed. Pharmaceuticallyacceptable antioxidants include, without limitation, sodiummetabisulfite, sodium thiosulfate, acetylcysteine, butylatedhydroxyanisole, and butylated hydroxytoluene. Useful preservativesinclude, without limitation, benzalkonium chloride, chlorobutanol,thimerosal, phenylmercuric acetate, phenylmercuric nitrate, a stabilizedoxy chloro composition, such as, e.g., sodium chlorite and chelants,such as, e.g., DTPA or DTPA-bisamide, calcium DTPA, andCaNaDTPA-bisamide. Tonicity adjustors useful in a pharmaceuticalcomposition include, without limitation, salts such as, e.g., sodiumchloride, potassium chloride, mannitol or glycerin and otherpharmaceutically acceptable tonicity adjustor. The pharmaceuticalcomposition may be provided as a salt and can be formed with many acids,including but not limited to, hydrochloric, sulfuric, acetic, lactic,tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueousor other protonic solvents than are the corresponding free base forms.It is understood that these and other substances known in the art ofpharmacology can be included in a pharmaceutical composition useful inthe invention.

Aspects of the present specification provide, in part, a method oftreating a soft tissue condition of an individual by administering acomposition disclosed herein. As used herein, the term “treating,”refers to reducing or eliminating in an individual a cosmetic orclinical symptom of a soft tissue condition characterized by a softtissue imperfection, defect, disease, and/or disorder; or delaying orpreventing in an individual the onset of a cosmetic or clinical symptomof a condition characterized by a soft tissue imperfection, defect,disease, and/or disorder. For example, the term “treating” can meanreducing a symptom of a condition characterized by a soft tissue defect,disease, and/or disorder by, e.g., at least 20%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90% or at least 100%. The effectiveness of a compound disclosed hereinin treating a condition characterized by a soft tissue defect, disease,and/or disorder can be determined by observing one or more cosmetic,clinical symptoms, and/or physiological indicators associated with thecondition. An improvement in a soft tissue defect, disease, and/ordisorder also can be indicated by a reduced need for a concurrenttherapy. Those of skill in the art will know the appropriate symptoms orindicators associated with specific soft tissue defect, disease, and/ordisorder and will know how to determine if an individual is a candidatefor treatment with a compound or composition disclosed herein.

A composition or compound is administered to an individual. Anindividual is typically a human being. Typically, any individual who isa candidate for a conventional soft tissue replacement procedure is acandidate for a soft tissue replacement procedure disclosed herein. Inaddition, the presently disclosed compositions and methods may apply toindividuals seeking a small/moderate enlargement, shape change orcontour alteration of a body part or region, which may not betechnically possible or aesthetically acceptable with existing softtissue implant technology. Pre-operative evaluation typically includesroutine history and physical examination in addition to thoroughinformed consent disclosing all relevant risks and benefits of theprocedure.

The composition and methods disclosed herein are useful in treating asoft tissue condition. A soft tissue condition includes, withoutlimitation, a soft tissue imperfection, defect, disease, and/ordisorder. Non-limiting examples of a soft tissue condition includebreast imperfection, defect, disease and/or disorder, such as, e.g., abreast augmentation, a breast reconstruction micromastia, thoracichypoplasia, Poland's syndrome, defects due to implant complications likecapsular contraction and/or rupture; a facial imperfection, defect,disease or disorder, such as, e.g., a facial augmentation, a facialreconstruction, Parry-Romberg syndrome, lupus erythematosus profundus,dermal divots, sunken checks, thin lips, nasal imperfections or defects,retro-orbital imperfections or defects, a facial fold, line and/orwrinkle like a glabellar line, a nasolabial line, a perioral line,and/or a marionette line, and/or other contour deformities orimperfections of the face; a neck imperfection, defect, disease ordisorder; a skin imperfection, defect, disease and/or disorder; othersoft tissue imperfections, defects, diseases and/or disorders, such as,e.g., an augmentation or a reconstruction of the upper arm, lower arm,hand, shoulder, back, torso including abdomen, buttocks, upper leg,lower leg including calves, foot including plantar fat pad, eye,genitals, or other body part, region or area, or a disease or disorderaffecting these body parts, regions or areas; urinary incontinence,fecal incontinence, other forms of incontinence; and gastroesophagealreflux disease (GERD).

The amount of adipose tissue used with any of the methods as disclosedherein will typically be determined based on the alteration and/orimprovement desired, the reduction and/or elimination of a soft tissuecondition symptom desired, the clinical and/or cosmetic effect desiredby the individual and/or physician, and the body part or region beingtreated. The effectiveness of adipose tissue administration may bemanifested by one or more of the following clinical and/or cosmeticmeasures: altered and/or improved soft tissue shape, altered and/orimproved soft tissue size, altered and/or improved soft tissue contour,altered and/or improved tissue function, improved transplant tissuesurvival, improved patient satisfaction and/or quality of life, anddecreased use of implantable foreign material.

For example, for breast augmentation procedures, effectiveness of thecompositions and methods may be manifested by one or more of thefollowing clinical and/or cosmetic measures: increased breast size,altered breast shape, altered breast contour, sustained engraftment,decreased rate of liponecrotic cyst formation, improved patientsatisfaction and/or quality of life, and decreased use of breastimplant.

As another example, effectiveness of the compositions and methods intreating a facial soft tissue may be manifested by one or more of thefollowing clinical and/or cosmetic measures: increased size, shape,and/or contour of facial feature like increased size, shape, and/orcontour of lip, cheek or eye region; altered size, shape, and/or contourof facial feature like altered size, shape, and/or contour of lip, cheekor eye region shape; reduction or elimination of a wrinkle, fold or linein the skin; resistance to a wrinkle, fold or line in the skin;rehydration of the skin; increased elasticity to the skin; reduction orelimination of skin roughness; increased and/or improved skin tautness;reduction or elimination of stretch lines or marks; increased and/orimproved skin tone, shine, brightness and/or radiance; increased and/orimproved skin color, reduction or elimination of skin paleness;sustained engraftment of composition; decreased side effects; improvedpatient satisfaction and/or quality of life.

As yet another example, for urinary incontinence procedures,effectiveness of the compositions and methods for sphincter support maybe manifested by one or more of the following clinical measures:decreased frequency of incontinence, sustained engraftment, improvedpatient satisfaction and/or quality of life, and decreased use ofimplantable foreign filler.

The amount of a compound used with any of the methods disclosed hereinwill typically be a therapeutically effective amount. As used herein,the term “therapeutically effective amount” is synonymous with“effective amount”, “therapeutically effective dose”, and/or “effectivedose” and refers to the amount of compound that will elicit thebiological, cosmetic or clinical response being sought by thepractitioner in an individual in need thereof. As a non-limitingexample, an effective amount is an amount sufficient to promoteformation of a blood supply sufficient to support the transplantedtissue. As another non-limiting example, an effective amount is anamount sufficient to promote formation of new blood vessels andassociated vasculature (angiogenesis) and/or an amount sufficient topromote repair or remodeling of existing blood vessels and associatedvasculature. The appropriate effective amount to be administered for aparticular application of the disclosed methods can be determined bythose skilled in the art, using the guidance provided herein. Forexample, an effective amount can be extrapolated from in vitro and invivo assays as described in the present specification. One skilled inthe art will recognize that the condition of the individual can bemonitored throughout the course of therapy and that the effective amountof a compound or composition disclosed herein that is administered canbe adjusted accordingly.

In aspects of this embodiment, the amount of a compound added is, e.g.,0.01 mg, 0.05 mg, 0.1 mg, 0.5 mg, 1 mg, 5 mg, 10 mg, 20 mg, 30 mg, 40mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg,500 mg, 750 mg, or 1000 mg. In other aspects of this embodiment, theamount of a compound added is, e.g., 0.01 mg/mL, 0.05 mg/mL, 0.1 mg/mL,0.5 mg/mL, 1 mg/mL, 5 mg/mL, 10 mg/mL, 20 mg/mL, 30 mg/mL, 40 mg/mL, 50mg/mL, 60 mg/mL, 70 mg/mL, 80 mg/mL, 90 mg/mL, 100 mg/mL, 150 mg/mL, 200mg/mL, 250 mg/mL, 500 mg/mL, 750 mg/mL, or 1000 mg/mL. In yet otheraspects of this embodiment, the amount of a compound added is, e.g.,0.01 mg/10 mL of tissue, 0.05 mg/10 mL of tissue, 0.1 mg/10 mL oftissue, 0.5 mg/10 mL of tissue, 1 mg/10 mL of tissue, 5 mg/10 mL oftissue, 10 mg/10 mL of tissue, 20 mg/10 mL of tissue, 30 mg/10 mL oftissue, 40 mg/10 mL of tissue, 50 mg/10 mL of tissue, 60 mg/10 mL oftissue, 70 mg/10 mL of tissue, 80 mg/10 mL of tissue, 90 mg/10 mL oftissue, 100 mg/10 mL of tissue, 150 mg/10 mL of tissue, 200 mg/10 mL oftissue, 250 mg/10 mL of tissue, 500 mg/10 mL of tissue, 750 mg/10 mL oftissue, or 1000 mg/10 mL of tissue.

In aspects of this embodiment, the amount of a compound added is, e.g.,about 0.01 mg to about 0.1 mg, about 0.1 mg to about 1 mg, about 1 mg toabout 10 mg, about 10 mg to about 100 mg, or about 100 mg to about 1000mg. In other aspects of this embodiment, the amount of a compound addedis, e.g., about 0.01 mg/mL to about 0.1 mg/mL, about 0.1 mg/mL to about1 mg/mL, about 1 mg/mL to about 10 mg/mL, about 10 mg/mL to about 100mg/mL, or about 100 mg/mL to about 1000 mg/mL. In yet other aspects ofthis embodiment, the amount of a compound added is, e.g., about 0.01mg/10 mL of tissue to about 0.1 mg/10 mL of tissue, about 0.1 mg/10 mLof tissue to about 1 mg/10 mL of tissue, about 1 mg/10 mL of tissue toabout 10 mg/10 mL of tissue, about 10 mg/10 mL of tissue to about 100mg/10 mL of tissue, or about 100 mg/10 mL of tissue to about 1000 mg/10mL of tissue.

In aspects of this embodiment, the amount of a compound added is, e.g.,about 0.0001% (w/v) to about 5% (w/v), about 0.001% (w/v) to about 5%(w/v), about 0.01% (w/v) to about 5% (w/v), about 0.1% (w/v) to about 5%(w/v), or about 1% (w/v) to about 5% (w/v). In other aspects of thisembodiment, the amount of a compound added is, e.g., about 0.001% (w/v)to about 1.0% (w/v), about 0.01% (w/v) to about 1.0% (w/v), or about0.1% (w/v) to about 1.0% (w/v).

The route of administration of composition administered to an individualpatient will typically be determined based on the cosmetic and/orclinical effect desired by the individual and/or physician and the bodypart or region being treated. Compositions or adipose tissue may beadministered by any means known to persons of ordinary skill in the artincluding, without limitation, syringe with needle, catheter, or bydirect surgical implantation. In addition, composition or adipose tissuecan be administered once, or over a plurality of times. Ultimately, thetiming used will follow quality care standards.

For a breast soft tissue replacement procedure, the route ofadministration may include axillary, periareolar, and/or inframammaryroutes. For a facial soft tissue replacement procedure, the route ofadministration can be frontal, temporal, zygomatic, periocular,mandibula, perioral or chin routes. In urinary incontinence procedures,the route of administration may include transurethral or periurethralroutes. Alternatively or in addition, the transplant may be deliveredvia an antegrade route. The routes discussed herein do not exclude theuse of multiple routes to achieve the desired clinical effect, orumbilical incision. Alternatively or in addition, cell-enhanced tissuemay be delivered through a transaxillary endoscopic subpectoralapproach.

A composition or compound administered to an individual to treat a softtissue condition promotes formation of a blood supply sufficient tosupport the transplanted tissue. Blood supply formation includes,without limitation, formation of new blood vessels and associatedvasculature (angiogenesis) and repair or remodel of existing bloodvessels and associated vasculature.

Aspects of the present specification disclose, in part, a method oftreating a soft tissue condition of an individual, the method comprisingthe steps of a) administering the adipose tissue to a site of the softtissue condition; and b) administering a composition comprising acompound as disclosed herein to the site of the soft tissue condition.The adipose tissue and compounds administered includes the adiposetissue disclosed herein. In these methods, the adipose tissue isadministered separately from a composition comprising the compound.Thus, in one aspect of this embodiment, adipose tissue is administeredfirst, following by administration of a composition comprising thecompound. In another aspect of this embodiment, a composition comprisinga compound is administered first, following by administration of adiposetissue.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments now contemplated. These examples should notbe construed to limit any of the embodiments described in the presentspecification, including those pertaining to the compositions andmethods of soft tissue replacement disclosed herein.

Example 1 Synthesis of Compounds of Formula I

This example illustrates the synthesis scheme for the compounds offormula I.

The methods of preparing the compounds disclosed herein are furtherillustrated by the following non-limiting examples, which are summarizedin the reaction schemes of FIGS. 1-9 wherein the compounds areidentified by the same designator in both the Examples and the Figures.

2-Alkyl-cyclopentane-1,3-dione (1a, FIG. 1)

A mixture of 1,3-cyclopentanedione (89.4 mmol, Aldrich), I-R² (96.4mmol, Aldrich), and KOH (5.097 g, 90.8 mmol) in water (25 mL)/dioxane(75 mL) was heated at reflux. After 5 hours, a solution of KOH (2 g) andI-R² (2 mmol) in water (5 mL)/dioxane (15 mL) was added and afteranother 3 hours at reflux, the solution was stirred at room temperatureovernight. A solution of KOH (2 g) and I-R² (2.4 mmol) in water (5mL)/dioxane (15 mL) was added to the overnight reaction and heating atreflux. After 4 hours, the mixture was cooled to room temperature andextracted with ether (1×100 mL, 3×75 mL). The combined ether extractswere evaporated, the residue combined with 10% HCl (50 mL), and theresulting mixture placed in a 120° C. oil bath until boiling wasobserved (ca. 15 minutes). The mixture was cooled to room temperature,neutralized by addition of NaHCO₃ solution (150 mL, saturated) and theresulting mixture extracted with CH₂Cl₂ (4×75 mL). The combined CH₂Cl₂solution was dried (MgSO₄), filtered and evaporated to leave a brown oilwhich was used directly in the next step.

2-Alkyl-2-methyl-cyclopentane-1,3-dione (2a, FIG. 1)

A mixture of 2-methyl-1,3-cyclopentanedione (10.025 g, 89.4 mmol,Aldrich), I-R² (96.4 mmol, Aldrich), and KOH (5.097 g, 90.8 mmol) inwater (25 mL)/dioxane (75 mL) was heated at reflux. After 5 hours, asolution of KOH (2 g) and I-R² (2 mmol) in water (5 mL)/dioxane (15 mL)was added and after another 3 hours at reflux, the solution was stirredovernight at room temperature. A solution of KOH (2 g) and I-R² (2.4mmol) in water (5 mL)/dioxane (15 mL) was added to the overnightreaction and heating at reflux. After 4 hours, the mixture was cooled toroom temperature and extracted with ether (1×100 mL, 3×75 mL). Thecombined ether extracts were evaporated, the residue combined with 10%HCl (50 mL), and the resulting mixture placed in a 120° C. oil bathuntil it began boiling (ca. 15 minutes). The mixture was cooled to roomtemperature, neutralized by addition of NaHCO₃ solution (150 mL,saturated) and the resulting mixture extracted with CH₂Cl₂ (4×75 mL).The combined CH₂Cl₂ solution was dried (MgSO₄), filtered and evaporatedto leave a brown oil which was used directly in the next step.

2,2-Dialkyl-methyl-cyclopentane-1,3-dione (2b, FIG. 1)

A mixture of 2-alkyl-1,3-cyclopentanedione 1a (89.4 mmol, Aldrich), I-R³(96.4 mmol, Aldrich), and KOH (5.097 g, 90.8 mmol) in water (25mL)/dioxane (75 mL) was heated at reflux. After 5 hours, a solution ofKOH (2 g) and I-R³ (2 mmol) in water (5 mL)/dioxane (15 mL) was addedand after another 3 hours at reflux, the solution was stirred at roomtemperature overnight. A solution of KOH (2 g) and I-R³ (2.4 mmol) inwater (5 mL)/dioxane (15 mL) was added to the overnight reaction andheating at reflux. After 4 hours, the mixture was cooled to roomtemperature and extracted with ether (1×100 mL, 3×75 mL). The combinedether extracts were evaporated, the residue combined with 10% HCl (50mL), and the resulting mixture placed in a 120° C. oil bath until itbegan boiling (ca. 15 minutes). The mixture was then cooled to roomtemperature, neutralized by addition of NaHCO₃ solution (150 mL,saturated) and the resulting mixture extracted with CH₂Cl₂ (4×75 mL).The combined CH₂Cl₂ solution was dried (MgSO₄), filtered and evaporatedto leave a brown oil which was used directly in the next step.

Spiro[2,4]heptane-4,7-dione (2c, FIG. 1)

A mixture of 2-alkyl-1,3-cyclopentanedione 1a (89.4 mmol, Aldrich),1,2-dibromoethane (120 mmol, Aldrich), and KOH (5.097 g, 90.8 mmol) inwater (25 mL)/dioxane (75 mL) was heated at reflux for 24 hours. Themixture was cooled, and the product extracted with ether (1×100 mL, 3×75mL). The combined ether extracts were evaporated, the residue combinedwith 10% HCl (50 mL), and the resulting mixture placed in a 120° C. oilbath until boiling was observed (ca. 15 minutes). The mixture was thencooled to room temperature, neutralized by addition of NaHCO₃ solution(150 mL, saturated) and the resulting mixture extracted with CH₂Cl₂(4×75 mL). The combined CH₂Cl₂ solution was dried (MgSO₄), filtered andevaporated to leave a brown oil which was used directly in the nextstep.

2,2-Dimethyl-cyclopentane-1,3-dione (2, FIG. 1)

Synthesized according to Agosta and Smith, J. Org. Chem. 35: 3856 (1970)A mixture of 2-methyl-1,3-cyclopentanedione (10.025 g, 89.4 mmol,Aldrich), methyl iodide (6.0 mL, 96.4 mmol, Aldrich), and KOH (5.097 g,90.8 mmol) in water (25 mL)/dioxane (75 mL) was heated at reflux. After5 hours, a solution of KOH (2 g) and MeI (2.4 mL) in water (5mL)/dioxane (15 mL) was added and after another 3 hours at reflux, thesolution was stirred at room temperature overnight. A solution of KOH (2g) and MeI (2.4 mL) in water (5 mL)/dioxane (15 mL) was added to theovernight reaction and heating at reflux. After 4 hours, the mixture wascooled to room temperature and extracted with ether (1×100 mL, 3×75 mL).The combined ether extracts were evaporated, the residue combined with10% HCl (50 mL), and the resulting mixture placed in a 120° C. oil bathuntil it began boiling (ca. 15 minutes). The mixture was cooled to roomtemperature, neutralized by addition of saturated NaHCO₃ solution (150mL) and the resulting mixture extracted with CH₂Cl₂ (4×75 mL). Thecombined CH₂Cl₂ solution was dried (MgSO₄), filtered and evaporated toleave a brown oil (10.474 g, 83 mmol, 93%) which was used directly inthe next step.

(S)-3-Hydroxy-2,2-dimethyl-cyclopentanone (3, FIG. 2)

Synthesized according to Brooks, et al., J. Org. Chem. 52: 3223 (1987).A 35° C. (internal temperature) solution of D-glucose (106.73 g, 592mmol, Aldrich) in water (690 mL) in a 4 L Erlenmeyer was treated withbaker's yeast (71.065 g, Fleischmann's). The mixture was fermented for 2hours, and 2,2-dimethyl-cyclopentane-1,3-dione (2) (7.316 g, 58 mmol)was added. The mixture was stirred for 48 hours and filtered throughcelite, washing with about 1 L CH₂Cl₂. About 100 mL of brine was addedto the filtrate and the layers separated using a separatory funnel.Brine (400 mL) was added to the aqueous layer and the resulting solutionextracted further with CH₂Cl₂ (3×500 mL). The combined CH₂Cl₂ solutionwas dried (MgSO₄), filtered and evaporated to leave a yellow oil. Flashchromatography (11×5 cm, 20% EtOAc/hexs→25%→30%→40%→>50%) gave alcohol 3(2.435 g, 19 mmol, 33%).

The enantiomeric excess of 3 was assayed by ¹H NMR of the correspondingMosher's ester which was prepared by treatment of alcohol 3 (11 mg, 0.09mmol) in dichloroethane (0.3 mL, Aldrich) with pyridine (27 μL, 0.33mmol, Aldrich) and (R)-α-methoxy-α-trifluoromethyphenylacetic acidchloride (58 μL, 0.31 mmol, Fluka). The mixture was stirred overnightand then partitioned between water (10 mL) and ether (10 mL). The etherlayer was washed with 1 M HCl (10 mL) and saturated NaHCO₃ solution andthen dried (MgSO₄), filtered and evaporated. ¹H NMR analysis was done onthe crude ester.

(S)-3-(tert)-Butyl-dimethyl-silanyloxy-2,2-dimethyl-cyclopentanone (4,FIG. 2)

A solution of alcohol (3) (520 mg, 4.1 mmol) and 2,6-lutidine (0.56 mL,4.8 mmol, Aldrich) in CH₂Cl₂ (8.0 mL, Aldrich) was treated with TBSOTf(1.0 mL, 4.3 mmol, Aldrich). After 5.5 hours, saturated NaHCO₃ solution(20 mL) was added and the mixture extracted with CH₂Cl₂ (20 mL). TheCH₂Cl₂ solution was washed with 20 mL each of 1 M HCl, saturated NaHCO₃solution, and brine and then dried (MgSO₄), filtered and evaporated.Flash chromatography (5×5 cm, 10% Et₂O/pentane) gave TBS ether (4) (698mg, 2.9 mmol, 70%).

(S)-3-(tert)-Butyl-dimethyl-silanyloxy-2,2-dimethyl-5-phenylselanyl-cyclopentanone(5, FIG. 2)

A solution of TBS ether (4) (1.496 g, 6.2 mmol) in THF (2 mL, Aldrich)was added dropwise to a −78° C. solution of LDA (4.9 mL, 7.3 mmol, 1.5M/cyclohexane, Aldrich) in THF (22 mL, Aldrich), rinsing with 2 mL THF.After 15 minutes, a solution of PhSeCl (1.424 g, 7.4 mmol, Aldrich) inTHF (2 mL) was quickly added by cannula, rinsing with 2 mL THF. Thesolution was stirred for 10 minutes and then partitioned between 50 mL0.5 M HCl and 75 mL ether. The ether layer was washed with 30 mL each ofwater, saturated NaHCO₃ solution, and brine and then dried (MgSO₄),filtered and evaporated. Flash chromatography (2% EtOAc/hexs→4%) gavephenylselenide (5) (1.641 g, 4.1 mmol, 67%) along with 476 mg of mixedfractions containing a lower R_(f) impurity.

(S)-4-(tert)-Butyl-dimethyl-silanyloxy-5,5-dimethyl-cyclopent-2-enone(6, FIG. 2)

A solution of selenide (5) (1.641 g, 4.1 mmol) and pyridine (0.62 mL,7.7 mmol, Aldrich) in CH₂Cl₂ (13 mL, Aldrich) was treated with water (1mL) and 30% H₂O₂ (1.1 mL, Aldrich). The mixture was stirred for 30minutes and then partitioned between 25 mL CH₂Cl₂ and 25 mL saturatedNaHCO₃ solution. The aqueous layer was extracted with 25 mL CH₂Cl₂ andthe combined CH₂Cl₂ solution washed with 1 M HCl (2×25 mL) and brine (50mL). The solution was dried (MgSO₄), filtered and evaporated to leave anorange oil. Flash chromatography (6×4 cm, 10% ether/pentane) gave enone(6) (572 mg, 2.4 mmol, 59%).

(3-Chloro-benzo[b]thiophen-2-yl)-methanol (12, FIG. 3)

To an ice cold solution of 10.0 g (47.0 mmol) of3-chloro-benzo[b]thiophene-2-carboxylic acid (11) in 200 mL of THF wasadded 47 mL of LiAlH₄ (47 mmol, 1 M/THF). After 3 hours, the reactionwas quenched by adding methanol (ca. 40 mL). The volatiles wereevaporated and the residue treated with 50 mL 1 M HCl. After stirringfor 10 minutes, the mixture was extracted with CH₂Cl₂ (3×150 mL). Thecombined CH₂Cl₂ solution was dried (MgSO₄), filtered and evaporated.Purification by flash chromatography on silica gel (10-20% ethylacetate/hexane) gave 4.32 g (21.6 mmol, 46%) of the alcohol (12).

3-Chloro-benzo[b]thiophene-2-carbaldehyde (13, FIG. 3)

A solution of alcohol 12 (4.32 g, 21.6 mmol) in 40 mL of CH₂Cl₂ wastreated with 4A molecular sieves, NMO (3.81 g, 32.5 mmol), and TPAP (381mg, 1.08 mmol). The reaction was stirred for 10 minutes and then driedby evaporated. Purification by flash chromatography on silica gel (2%ethyl acetate/hexane) gave 3.52 g (18.3 mmol, 84%) of the aldehyde (13).

(E)-3-(3-Chloro-benzo[b]thiophen-2-yl)-acrylic acid methyl ester (14,FIG. 3)

A solution of 3.52 g (18.3 mmol) of (13) in 50 mL toluene was treatedwith methyl(triphenylphosphoranylidene)acetate (7.48 g, 21.9 mmol).After 4 hours, saturated NaHCO₃ solution (50 mL) was added and themixture extracted with ethyl acetate (2×75 mL). The combined ethylacetate solution was washed with brine (50 mL), dried (Na₂SO₄), filteredand evaporated. Purification by flash chromatography on silica gel (5%ethyl acetate/hexane) provided 3.60 g (14.6 mmol, 80%) of the enoate(14).

3-(3-Chloro-benzo[b]thiophen-2-yl)-propionic acid methyl ester (15, FIG.3)

A solution of 3.60 g (14.6 mmol) of (14) in 50 mL THF was treated withWilkinson's catalyst (3.35 g, 3.62 mmol). The mixture was stirred under1 atm H₂ for 18 hours and then was filtered through celite. The solventwas evaporated and the residue purified by flash chromatography onsilica gel (0-2% ethyl acetate/hexane) to give 3.63 g (14.3 mmol, 99%)of the saturated ester (15).

3-(3-Chloro-benzo[b]thiophen-2-yl)-propan-1-ol (16, FIG. 3)

An ice cold solution of 3.63 g (14.3 mmol) of (15) in 60 mL of ether wastreated with LiBH₄ (621 mg, 28.5 mmol) and methanol (2 mL). After 30minutes, 30 mL of 0.5 M NaOH solution was added. The mixture wasextracted with ethyl acetate (2×25 mL) and the combined ethyl acetatesolution was washed with brine (50 mL), dried (MgSO₄), filtered andevaporated. The residue was purified by flash chromatography on silicagel (5-20% ethyl acetate/hexane) to give 2.57 g (11.3 mmol, 79%) of thealcohol (16).

3-(3-Chloro-benzo[b]thiophen-2-yl)-propionaldehyde (17, FIG. 3)

A −78° C. solution of oxalyl chloride (1.73 g, 13.6 mmol) indichloromethane (20 mL) was treated with DMSO (20 mL). After 5 minutes,a solution of alcohol (16) (2.57 g, 11.3 mmol) in dichloromethane (20mL) was added. After another 15 minutes, triethylamine (7.1 mL, 50.6mmol) was added. The reaction was stirred at −78° C. for 5 minutes, andwarmed to room temperature. After 30 minutes, 100 mL water was added andthe mixture extracted with dichloromethane (3×60 mL). The combineddichloromethane solution was dried (Na₂SO₄), filtered and evaporated.Purification by flash chromatography on silica gel (10% ethylacetate/hexane) gave 2.11 g (9.4 mmol, 83%) of the aldehyde (17).

5-(3-Chloro-benzo[b]thiophen-2-yl)-pent-1-yn-3-ol (18, FIG. 3)

A solution of aldehyde (17) (2.11 g, 9.4 mmol) in 15 mL THF was added toa solution of ethynylmagnesium bromide (28.2 mL, 14.1 mmol, 0.5 M THF)at 0° C. After 1.5 hours, saturated NH₄Cl solution (75 mL) was added andthe mixture was extracted with ethyl acetate (3×50 mL). The combinedethyl acetate solution was washed with brine (50 mL) and then dried(Na₂SO₄), filtered and evaporated. Purification by flash chromatography(5-20% ethyl acetate/hexane) gave 2.20 g (8.78 mmol, 93%) of the alcohol(18).

tert-Butyl-{1-[2-(3-chloro-benzo[b]thiophen-2-yl)-ethyl]-prop-2-ynyloxy}-dimethyl-silane(19, FIG. 3)

A solution of alcohol (18) (2.20 g, 8.78 mmol) in dichloromethane (15mL) was treated with DMAP (215 mg, 1.8 mmol), TBSCl (1.59 g, 10.5 mmol),and triethylamine (1.8 mL, 13.2 mmol). The reaction was stirred for 24hours and then saturated sodium bicarbonate solution (50 mL) was added.The mixture was extracted with dichloromethane (2×50 mL) and thecombined dichloromethane solution dried (Na₂SO₄), filtered andevaporated. Purification by flash chromatography (4% ethylacetate/hexane) gave 3.06 g (6.4 mmol, 73%) of the protected alcohol(19).

tert-Butyl-hex-5-ynyloxy-dimethyl-silane (26);7-(tert-Butyl-dimethyl-silanyloxy)-hept-2-yn-1-ol (27); and Acetic acid7-(tert-butyl-dimethyl-silanyloxy)-hept-2-ynyl ester (28, FIG. 4)

A solution of 7-(tert-Butyl-dimethyl-silanyloxy)-hept-2-yn-1-ol (27)(4.507 g, 21 mmol) in pyridine (20 mL) was treated with acetic anhydride(3.0 mL, 31.8 mmol). After 18 hours, the solvent was evaporated and theresidue co-evaporated with toluene. The residue was used directly in thenext step.

7-Acetoxy-hept-5-ynoic acid (29, FIG. 4)

A solution of crude (28) in acetone (100 mL) was treated with JonesReagent (18.0 mL, 41.4 mmol, 2.3 M) and cooled with an ice bath. After 1hour at room temperature, 10 mL isopropyl alcohol was added and themixture stirred for 15 minutes. The mixture still had a brown color soanother 10 mL isopropyl alcohol was added. After another 15 minutes, thecolor had not changed so the mixture was filtered through celite and thefiltrate evaporated in vacuo. The residue was partitioned between 100 mLether and 100 mL saturated ammonium chloride solution. The aqueous layerwas extracted with 100 mL ether and the combined ether solution washedwith brine and then dried (MgSO₄), filtered and evaporated to leave ayellow oil (6.333 g) that was used directly in the next step.

7-Hydroxy-hept-5-ynoic acid methyl ester (30, FIG. 4)

The crude acid (29) (6.333 g) was treated with a 1% solution of acetylchloride in methanol (60 mL). After 16 hours, sodium bicarbonate (1.966g, 23.4 mmol) was added. The mixture was dried (MgSO₄), filtered throughcelite and evaporated in vacuo. Purification by flash chromatography onsilica gel (30-40% ethyl acetate/hexanes) gave 7-Hydroxy-hept-5-ynoicacid methyl ester (30) (3.022 g, 19.3 mmol, 92% from7-(tert-Butyl-dimethyl-silanyloxy)-hept-2-yn-1-ol (27).

7-Iodo-hept-5-ynoic acid methyl ester (31, FIG. 4)

A solution of (30) (1.347 g, 8.6 mmol) in 5 mL dichloromethane was addedto a mixture of triphenylphosphine (2.725 g, 10.4 mmol), imidazole (726mg, 10.7 mmol), and iodine (2.602 g, 10.3 mmol) in 34 mLdichloromethane, rinsing with 5 mL dichloromethane. After 40 minutes,the dichloromethane was evaporated in vacuo to about 2 mL and theresulting mixture filtered through basic alumina, washing with 10% ethylacetate/hexanes. Purification by flash chromatography on silica gel (10%ethyl acetate/hexanes) gave 1.878 g (7.1 mmol, 83%) of the propargyliodide.

tert-Butyl-{(E)-1-[2-(3-chloro-benzo[b]thiophen-2-yl)-ethyl]-3-iodo-allyloxy}-dimethyl-silane(32, FIG. 5)

A solution of alkyne (19) (5.547 g, 15.2 mmol) in dichloromethane (50mL) was treated with Cp₂ZrHCl (5.794 g, 22.5 mmol). The reaction wasstirred for 45 minutes and then N-iodosuccinimide (4.966 g, 22.1 mmol)was added. After 15 minutes, saturated sodium bicarbonate solution (200mL) was added and the mixture extracted with dichloromethane (2×100 mL).The combined dichloromethane solution was dried (MgSO₄), filtered andevaporated. Purification by flash chromatography on silica gel (0-5%ethyl acetate/hexanes) gave 6.608 g (13.1 mmol, 86%) of the vinyl iodide(32).

7-{(1R,4S,5R)-4-(tert-Butyl-dimethyl-silanyloxy)-5-[(E)-3-(tert-butyl-dimethyl-silanyloxy)-5-(3-chloro-benzo[b]thiophen-2-yl)-pent-1-enyl]-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-ynoicacid methyl ester (33, FIG. 5)

A −78° C. solution of iodide (32) (675 mg, 1.34 mmol) in THF (2.0 mL)was treated with tert-butyllithium (1.73 mL, 2.94 mL, 1.7 M/pentane).The dark red mixture was stirred for 25 minutes and then dimethylzinc(0.80 mL, 1.6 mmol, 2 M/toluene) was added. The solution was stirred at0° C. for 15 minutes and then recooled to −78° C. A solution of enone(6) (208 mg, 0.87 mmol) in THF (1.0 mL) was added over 2 hours bysyringe pump, rinsing with 0.5 mL THF. After 30 minutes, HMPA (1.34 mL,distilled from CaH₂) was added followed by a solution of propargyliodide (31) (1.286 g, 4.83 mmol) in THF (1.0 mL). The solution wasstirred in a −40° C. bath overnight and then 20 mL saturated ammoniumchloride solution and 10 mL water were added. The mixture was extractedwith dichloromethane (20 mL) and ethyl acetate (2×20 mL). The combinedorganic extracts were dried (MgSO₄), filtered and evaporated.Purification by flash chromatography on silica gel (5-10% ethylacetate/hexanes) gave 198 mg (0.27 mmol, 31%) of (33).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-ynoicacid methyl ester (34, 35, FIG. 5)

A solution of (33) (198 mg, 0.27 mmol) in CH₃CN (6.5 mL) was treatedwith HF-pyridine (1.2 mL). The solution was stirred for 3 hours andsaturated sodium bicarbonate solution (120 mL) was added. The mixturewas extracted with dichloromethane (3×50 mL) and the combineddichloromethane solution dried (Na₂SO₄), filtered and evaporated.Purification by flash chromatography (50% ethyl acetate/hexane) followedby preparative TLC (55% ethyl acetate/hexane) gave 55 mg (0.11 mmol,41%) of the less polar diastereomer (34) and 51 mg (0.10 mmol, 37%) ofthe more polar diastereomer (35).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-ynoicacid (low R_(f) diastereomer, 36, FIG. 5)

A solution of (34) (9 mg, 0.017 mmol) and rabbit liver esterase (1 mg)in pH 7.2 phosphate buffer (2 mL)/CH₃CN (0.1 mL) was stirred for 17hours. The mixture was then coevaporated with CH₃CN to remove water andthe residue purified by flash chromatography on silica gel (3-7%MeOH/CH₂Cl₂) to give 8 mg (0.016 mmol, 93%) of the acid (36).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-ynoicacid (high R_(f) diastereomer, 37, FIG. 5)

A solution of (35) (12 mg, 0.023 mmol) and rabbit liver esterase (1 mg)in pH 7.2 phosphate buffer (2 mL)/CH₃CN (0.1 mL) was stirred for 17hours. TLC showed the presence of starting material, so another 2 mg ofthe esterase was added. After stirring for another 24 hours, thereaction was complete. Work up and purification as above for (36) gave 8mg (0.016 mmol, 69%) of the acid (37).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoicacid methyl ester (low R_(f) diastereomer, 38, FIG. 5)

Ethanol (95%, 2.5 mL) was added to NiCl₂ (50 mg, 0.39 mmol) and NaBH₄ (7mg, 0.19 mmol). The resulting black mixture was stirred for 5 minutesand then ethylenediamine (41 μL, 0.61 mmol) was added. After 15 minutes,a solution of alkyne (34) (40 mg, 0.077 mmol) in 0.5 mL 95% ethanol wasadded, rinsing with 0.5 mL ethanol. The flask was purged with H₂ andallowed to stir under 1 atm H₂ for 22 hours. The mixture was thenfiltered through celite and purified by flash chromatography on silicagel (55% ethyl acetate/hexanes) to give 17 mg (0.032 mmol, 43%) of thealkene (38).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoicacid methyl ester (high R_(f) diastereomer 39, FIG. 5)

The same procedure as for (36) was followed to give 17 mg (0.032 mmol,41%) of (39).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoicacid (low R_(f) diastereomer, 40, FIG. 5)

The same procedure as above for (36) was used to give 9 mg (0.018 mmol,85%) of acid (40). 300 MHz ¹H NMR (CDCl₃, ppm) δ 7.73 (2H, d, J=8.4 Hz)7.45-7.30 (2H, m) 5.8-5.6 (2H, m) 5.4-5.3 (2H, m) 4.3-4.1 (1H, m) 3.57(1H, d, J=9.7 Hz) 3.1-2.9 (2H, m) 2.5-1.9 (10H, m) 1.7-1.6 (2H, m) 1.09(3H, s) 0.89 (3H, s).

(Z)-7-{(1R,4S,5R)-5-[(E)-5-(3-chloro-benzo[b]thiophene-2-yl)-3-hydroxy-pent-1-enyl]-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl}-hept-5-enoicacid (high R_(f) diastereomer, 41, FIG. 5)

The same procedure as above for the (36) was used to give 9 mg (0.018mmol, 85%) of acid (41). 300 MHz ¹H NMR (CDCl₃, ppm) δ 7.73 (2H, d,J=8.8 Hz) 7.45-7.30 (2H, m) 5.8-5.6 (2H, m) 5.45-5.30 (2H, m) 4.3-4.2(1H, m) 3.61 (1H, d, J=9.7 Hz) 3.1-3.0 (2H, m) 2.5-1.9 (10H, m) 1.7-1.6(2H, m) 1.10 (3H, s) 0.90 (3H, s).

2-Benzo[b]thiophen-2-yl-ethanol (54b, FIG. 6)

n-BuLi (100 mL, 160 mmol, 1.6M/hexanes) was added to a −78° C. mixtureof thianaphthene (54a) (17.31 g, 129 mmol) in THF (70 mL)/ether (30 mL).The mixture was stirred at −78° C. for 2 hours and then a solution ofethylene oxide (42.86 g, 1.071 mmol) in THF (70 mL)/ether (30 mL) wasadded by cannula over 15 minutes. The resulting mixture was stirred for2 hours at −78° C. and then at room temperature for 15 hours. Themixture was evaporated, 200 mL water was added, and the resultingmixture was extracted with ethyl acetate (3×150 mL). The combinedorganic solution was washed with brine and then dried (Na₂SO₄),filtered, and evaporated. Purification by flash chromatography on silicagel (20% ethyl acetate/hexanes) gave (54b) (13.61 g, 78 mmol, 60%).

Benzo[b]thiophen-2-yl-acetaldehyde (54c, FIG. 6)

A 0° C. mixture of (54b) (8.019 g, 44.9 mmol) in 100 mL dichloromethanewas treated with Dess-Martin reagent (20 g, 47.2 mmol). The mixture wasstirred at 0° C. for 10 minutes and at room temperature for 40 minutes.Saturated NaHCO₃ solution (200 mL) and 0.1 M NaHSO₃ solution were addedand the resulting mixture was extracted with ethyl acetate (3×300 mL).The combined organic solution was dried (Na₂SO₄), filtered andevaporated to give (54c) (8.77 g). The aldehyde was taken on crude forthe next reaction.

1-Benzo[b]thiophen-2-yl-but-3-yn-2-ol (54d, FIG. 6)

A solution of crude (54c) (8.77 g) in THF (100 mL) was added to asolution of ethynylmagnesium bromide (450 mL, 225 mmol, 0.5 M/THF) at 0°C. by cannula. The mixture was stirred for 1 hour at 0° C. and for 1hour at room temperature. The reaction was then quenched by addition of200 mL saturated NH₄Cl solution. The layers were separated and theaqueous layer extracted with ethyl acetate (3×200 mL). The combinedorganic solution was washed with brine and then dried (Na₂SO₄), filteredand evaporated. Purification by flash chromatography on silica gel (10%20% ethyl acetate/hexanes) gave (54d) (7.67 g, 37.9 mmol, 84% from 5-2).

(1-Benzo[b]thiophen-2-ylmethyl-prop-2-ynyloxy)-tert-butyl-dimethyl-silane(54e, FIG. 6)

DMAP (2.306 g, 18.9 mmol), TBSCl (11.502 g, 76.3 mmol) and triethylamine(5.25 mL, 37.7 mmol) were added to a solution of (54d) (7.67 g, 37.9mmol) in dichloromethane (120 mL). After 17 hours, 150 mL of saturatedNH₄Cl solution was added and the layers were separated. The aqueouslayer was extracted with dichloromethane (3×100 mL) and the combinedorganic solution was dried (Na₂SO₄), filtered and evaporated.Purification by flash chromatography on silica gel (4% ethylacetate/hexanes) gave (54e) (8.38 g, 26.5 mmol, 70%).

((E)-1-Benzo[b]thiophen-2-ylmethyl-3-iodo-allyloxy)-tert-butyl-dimethyl-silane(54f, FIG. 6)

Cp₂ZrHCl (1.719 g, 6.67 mmol) was added to a solution of (54e) (1.372 g,4.34 mmol) in dichloromethane (30 mL). The reaction was stirred for 30minutes at room temperature and N-iodosuccinimide (1.997 g, 8.88 mmol)was added. After 1 hour, the reaction was poured into 100 mL ofsaturated NaHCO₃ solution. The resulting mixture was extracted withdichloromethane (3×75 mL) and the combined organic extracts dried(Na₂SO₄), filtered and evaporated. Purification by flash chromatographyon silica gel (2% ethyl acetate/hexanes) gave (54f) (1.7484 g, 91%).

(Z)-7-[(1R,4S,5R)-5-(E)-4-Benzo[b]thiophen-2-yl-3-hydroxy-but-1-enyl)-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl]-hept-5-enoicacid (54, 55, FIG. 6)

A solution of (54f) was treated in a manner similar to the schemeoutlined in FIG. 5 to produce (54) and (55).

(Z)-7-[(1R,4S,5R)-5-(E)-4-Benzo[b]thiophen-2-yl-3-hydroxy-but-1-enyl)-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl]-hept-5-enoicacid (2-hydroxy-ethyl)-amide (60-63, 65, FIG. 7)

A solution of acid (55) (7 mg, 0.015 mmol) in DMF (0.5 mL) was treatedwith N-hydroxysuccinimide (6.9 mg, 0.056 mmol). The mixture was stirredfor 5 minutes and 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimidehydrochloride (EDCl, 20.7 mg, 0.11 mmol) was added. After stirring for 7hours, 2-aminoethanol (5 μL, 0.083 mmol) was added and the mixturestirred further for 16 hours. Ethyl acetate (50 mL) was added and themixture washed with water (3×50 mL) and brine (50 mL). The organic layerwas dried (Na₂SO₄), filtered and evaporated. Purification by flashchromatography on silica gel (5% methanol/dichloromethane) followed bypreparative thin layer chromatography (10% methanol/dichloromethane)gave amide (65) (5 mg, 0.010 mmol, 65%). Amides (60-63) were prepared ina similar manner.

(Z)-7-[(1R,4S,5R)-5-(E)-4-Benzo[b]thiophen-2-yl-3-hydroxy-but-1-enyl)-4-hydroxy-3,3-dimethyl-2-oxo-cyclopentyl]-hept-5-enoicacid amide (69, FIG. 7)

A solution of acid (55) (9 mg, 0.02 mmol) in dichloromethane (0.2 mL)was treated with triethylamine (15 μL, 0.11 mmol). The solution wascooled to 0° C. and after 10 minutes, ethyl chloroformate (7 μL, 0.073mmol) was added. The solution was stirred further for 1 hour at 0° C.and then concentrated aqueous ammonium hydroxide solution was added (10μL, 0.26 mmol). The reaction was stirred at room temperature overnightand then quenched by addition of 0.5 M HCl (7 mL). The mixture wasextracted with ethyl acetate (3×30 mL), the combined ethyl acetatesolution washed with saturated NaHCO₃ solution (20 mL) and brine (20mL), and then dried (Na₂SO₄), filtered and evaporated. Purification byflash chromatography on silica gel (2%-6% methanol/dichloromethane) gavethe title amide (2.6 mg, 28%).

Example 2

Compounds of Formula I Promote Synthesis of TGF-β and VEGF

This example illustrates that compounds of formula I promote formationof blood vessels.

To test whether the compounds of Formula I promote the formation ofblood vessels, and incisional wound model was employed over a 14-dayperiod. Sprague-Dawley rats (180-200 g) were anesthetized, the backsshaved, and a 2-cm long incision was made on the back skin of rats understerile conditions, reaching the deep fascia. The wound was immediatelyclosed with 4-0 sutures. The animals were topically treated with vehicleor Compound 1 at 0.004% twice daily. The vehicle contains ethanol 30%,propylene glycol 12%, dipropylene glycol 5%, benzyl alcohol 5%, glycerol3% and normal saline 45%. Biopsy samples of skin wound tissues wereanalyzed with Western blots day-7 and day-14 post-surgery. Tissuesamples about 1 mm thick were taken from both sides of the wound.Protein was extracted, and FGF-2/VEGF expression was monitored, withWestern blot analysis.

Compound 1 treatment significantly enhanced the expression of FGF-2 by60%, as normalized with β-actin (a house-keeping gene) by day-7 of theexperiment (P=0.004). This significantly different increase in FGF-2synthesis persisted for the whole study period (P=0.04). VEGF expressionincreased 25% as normalized with β-actin, by Compound 1 treatment onday-7 (P=0.039). This significantly different increase is synthesisreturned to the baseline by day-14. Such an increase in FGF-2 and VEGFis consistent with their role in new blood vessel formation. Theup-regulation of FGF-2 and VEGF by Compound 1 treatment was alsoobserved in studies using an oral ulcer wound model.

Example 3 Adipose Tissue Transplant for Breast Defect Correction

This example illustrates the use of compositions and methods disclosedherein for a breast defect correction.

A 32-year-old woman presented with complaints that the medial portionsof her breast implants were visible, which accentuated the “bony”appearance of her sternum. In addition she felt her breast were too farapart. Pre-operative evaluation of the person includes routine historyand physical examination in addition to thorough informed consentdisclosing all relevant risks and benefits of the procedure. Thephysician evaluating the individual determines that she is a candidatefor a soft tissue replacement method using the compositions and methodsdisclosed herein.

To begin the procedure, adipose tissue is harvested from the woman. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the lateral and medial thigh regions.The harvested area is injected subcutaneously with a standard tumescentfluid solution containing a saline solution, 0.5% lidocaine, and about0.001% epinephrine. Using an 11-blade scalpel, a small puncture wound ismade in order to transverse the dermis. The blade is turned 360 degreesto complete the wound. A two-holed blunt harvesting cannula (3 mm innerdiameter) connected to a vacuum pump at low negative pressure (0.5 atm)is then inserted into the subcutaneous adipose tissue plane. The cannulais then moved throughout the plane to disrupt the connective tissuearchitecture. The volume of aspirate obtained is about 300 mL. Theharvest adipose tissue is processed by centrifugation at 3,000 g for 3minutes to separate health adipocytes and regenerative cells from blood,infiltration fluid and cell debris.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with the processed adiposetissue. The amount of compound added is an amount sufficient to promoteformation of a blood supply sufficient to support the transplantedtissue. This composition is then transferred to 3 mL syringes. One-holedblunt infiltration cannulas (3 mm inner diameter) are used to place theadipose tissue subcutaneously over the lateral sternum and medial breastbilaterally, 70 mL on the right and 50 mL on the left. The adiposetissue is administered in a tear like fashion to increase the surfacearea to volume ratio.

Alternatively, the adipose tissue is first administered into theindividual, and a compound of formula I, such as, e.g., Compound 1,Compound 2, Compound 3, Compound 4, and/or Compound 5, is subsequentlyadministered into the same, or in the vicinity of, the region where theadipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure. Approximately one month after the procedure,the woman indicates that his quality of life has improved.

Example 4 Adipose Tissue Transplant for Breast Augmentation

This example illustrates the use of compositions and methods disclosedherein for a breast augmentation.

A 28-year-old woman presented micromastia or breast hypoplasia.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for a softtissue replacement method using the compositions and methods disclosedherein.

To begin the procedure, adipose tissue is harvested from the woman. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the lateral and medial thigh regions.Using a 10-blade scalpel, a small puncture wound is made in order totransverse the dermis. The blade is turned 360 degrees to complete thewound. A two-holed blunt harvesting cannula (3 mm inner diameter)connected to a syringe is then inserted into the subcutaneous adiposetissue plane. The cannula is then moved throughout the plane to disruptthe connective tissue architecture. The volume of aspirate obtained isabout 600 mL. The harvest adipose tissue is processed by centrifugationat 2,700 g for 5 minutes to separate healthy adipocytes and regenerativecells from blood, infiltration fluid and cell debris. The centrifugedadipose tissue is then washed once is a Ringer's saline solution withlactone.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with the processed adiposetissue. The amount of compound added is an amount sufficient to promoteformation of a blood supply sufficient to support the transplantedtissue. This composition is then transferred to 10 mL syringes.One-holed blunt infiltration cannulas (3 mm inner diameter) are used toplace the adipose tissue subcutaneously using axillary, periareolar, andinframammary routes bilaterally, 190 mL on the right and 245 mL on theleft. The adipose tissue is administered in a tear like fashion toincrease the surface area to volume ratio.

Alternatively, the adipose tissue is first administered into theindividual, and a compound of formula I, such as, e.g., Compound 1,Compound 2, Compound 3, Compound 4, and/or Compound 5, is subsequentlyadministered into the same, or in the vicinity of, the region where theadipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure. Approximately one month after the procedure,the woman indicates that his quality of life has improved.

Example 5 Adipose Tissue Transplant for Breast Disorder

This example illustrates the use of compositions and methods disclosedherein for a breast disorder.

A 29-year-old woman presented with bilateral tuberous breast deformity.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for a softtissue replacement method using the compositions and methods disclosedherein.

To begin the procedure, adipose tissue is harvested from the woman. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the abdomen, buttock, lateral andmedial thigh, and trochanter regions. Using a 12-blade scalpel, a smallpuncture wound is made in order to transverse the dermis. The blade isturned 360 degrees to complete the wound. A two-holed blunt harvestingcannula (3 mm inner diameter) connected to a syringe is then insertedinto the subcutaneous adipose tissue plane. The cannula is then movedthroughout the plane to disrupt the connective tissue architecture. Thevolume of aspirate obtained is about 1,400 mL.

The harvested adipose tissue is divided into two, approximately equalportions. One portion is processed by gravity sedimentation to separatehealth adipocytes and regenerative cells from blood, infiltration fluidand cell debris. The other portion is used to isolate regenerativecells. This portion is digested with 0.075% collagenase in bufferedsaline for 30 minutes on a shaker at 37° C. Regenerative cells are thenseparated from mature adipocytes and connective tissue by centrifugingat 800 g for 10 minutes. The pellet containing the regenerative cells isthen washed three times with buffered saline. The washed regenerativecells are then added back to the sediment purified adipose tissue.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with the processed adiposetissue. The amount of compound added is an amount sufficient to promoteformation of a blood supply sufficient to support the transplantedtissue. This composition is then transferred to 10 mL syringes.One-holed blunt infiltration cannulas (3 mm inner diameter) are used toplace the adipose tissue subcutaneously in multiple planes axillary,periareolar, and inframammary routes bilaterally, 380 mL on the rightand 370 mL on the left. The adipose tissue is administered in a tearlike fashion to increase the surface area to volume ratio.

Alternatively, the adipose tissue is first administered into theindividual, and a compound of formula I, such as, e.g., Compound 1,Compound 2, Compound 3, Compound 4, and/or Compound 5, is subsequentlyadministered into the same, or in the vicinity of, the region where theadipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure. Approximately one month after the procedure,the woman indicates that his quality of life has improved.

Example 6 Adipose Tissue Transplant for Facial Defects of Check

This example illustrates the use of compositions and methods disclosedherein for a facial disorder.

A 28-year-old woman presented with a lean face. She felt her face lookedold, sad and bitter because of the less fullness of her cheek contour.Pre-operative evaluation of the person includes routine history andphysical examination in addition to thorough informed consent disclosingall relevant risks and benefits of the procedure. The physicianevaluating the individual determines that she is a candidate for a softtissue replacement method using the compositions and methods disclosedherein.

To begin the procedure, adipose tissue is harvested from the woman. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the abdominal region. The harvestedarea is injected subcutaneously with a standard tumescent fluid solutioncontaining a saline solution, 0.08% lidocaine, and about 0.001%epinephrine. Using an 11-blade scalpel, a small puncture wound is madein order to transverse the dermis. The blade is turned 360 degrees tocomplete the wound. A two-holed blunt harvesting cannula (2.5 mm innerdiameter) connected to a 60 mL syringe is then inserted into thesubcutaneous adipose tissue plane. The cannula is then moved throughoutthe plane to disrupt the connective tissue architecture. The volume ofaspirate obtained is about 50 mL. The harvest adipose tissue isprocessed by washing the harvested tissue with saline to removelidocaine, oil and residual blood. The washed tissue is thencentrifugation at 100 g for 2 minutes to separate health adipocytes andregenerative cells from any remaining blood, infiltration fluid and celldebris.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with the processed adiposetissue. The amount of compound added is an amount sufficient to promoteformation of a blood supply sufficient to support the transplantedtissue. This composition is then transferred to 20 mL syringes.One-holed blunt infiltration cannulas (3 mm inner diameter) are used toplace about 15 mL of adipose tissue subcutaneously and under superficialmusculoaponeurotic system into the left and right checks.

Alternatively, the adipose tissue is first administered into theindividual, and a compound of formula I, such as, e.g., Compound 1,Compound 2, Compound 3, Compound 4, and/or Compound 5, is subsequentlyadministered into the same, or in the vicinity of, the region where theadipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure because she looked younger. Approximately onemonth after the procedure, the woman indicates that his quality of lifehas improved.

Example 7 Adipose Tissue Transplant for Facial Defects of Eyelids

This example illustrates the use of compositions and methods disclosedherein for a facial disorder.

A 37-year-old woman presented with sunken eyes and this appearance madeher look old and fierce. Pre-operative evaluation of the person includesroutine history and physical examination in addition to thoroughinformed consent disclosing all relevant risks and benefits of theprocedure. The physician evaluating the individual determines that sheis a candidate for a soft tissue replacement method using thecompositions and methods disclosed herein.

To adipose tissue is harvested from the woman as described in Example 6.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with the processed adiposetissue. The amount of compound added is an amount sufficient to promoteformation of a blood supply sufficient to support the transplantedtissue. This composition is then transferred to 20 mL syringes.One-holed blunt infiltration cannulas (3 mm inner diameter) are used toplace about 2.5 mL of adipose tissue subcutaneously and undersuperficial musculoaponeurotic system into the upper eyelid regions.

Alternatively, the adipose tissue is first administered into theindividual, and a compound of formula I, such as, e.g., Compound 1,Compound 2, Compound 3, Compound 4, and/or Compound 5, is subsequentlyadministered into the same, or in the vicinity of, the region where theadipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure because she looked younger. Approximately onemonth after the procedure, the woman indicates that her quality of lifehas improved.

Example 8 Adipose Tissue Transplant for Facial Defects

This example illustrates the use of compositions and methods disclosedherein for a breast disorder.

A 33-year-old woman presented with depressed bilateral temporal andcheek areas. Pre-operative evaluation of the person includes routinehistory and physical examination in addition to thorough informedconsent disclosing all relevant risks and benefits of the procedure. Thephysician evaluating the individual determines that she is a candidatefor a soft tissue replacement method using the compositions and methodsdisclosed herein.

The adipose tissue is harvested from the woman as described in Example6, except that 200 mL of adipose tissue is collected.

The harvested adipose tissue is divided into two, approximately equalportions. One portion is processed by saline washing and centrifugationas described in Example 6. The other portion is used to isolateregenerative cells. This portion is digested with 0.075% collagenase inbuffered saline for 30 minutes on a shaker at 37° C. Regenerative cellsare then separated from mature adipocytes and connective tissue bycentrifuging at 800 g for 10 minutes. The pellet containing theregenerative cells is then washed three times with buffered saline. Thewashed regenerative cells are then added back to the purified adiposetissue.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with the processed adiposetissue. The amount of compound added is an amount sufficient to promoteformation of a blood supply sufficient to support the transplantedtissue. This composition is then transferred to 10 mL syringes.One-holed blunt infiltration cannulas (3 mm inner diameter) are used toplace about 16 mL of adipose tissue subcutaneously and under superficialmusculoaponeurotic system into the left and right temporal and cheeksregions.

Alternatively, the adipose tissue is first administered into theindividual, and a compound of formula I, such as, e.g., Compound 1,Compound 2, Compound 3, Compound 4, and/or Compound 5, is subsequentlyadministered into the same, or in the vicinity of, the region where theadipose tissue was implanted.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure because she looked younger. Approximately onemonth after the procedure, the woman indicates that her quality of lifehas improved.

Example 9 Adipose Tissue Transplant to Treat Stress Urinary Incontinence

This example illustrates the use of compositions and methods disclosedherein for treating stress urinary incontinence.

A 55 year old man presents with urinary incontinence. Pre-operativeevaluation of the patient includes routine history and physicalexamination in addition to thorough informed consent disclosing allrelevant risks and benefits of the procedure. The physician evaluatingthe individual determines that he is a candidate for a soft tissuereplacement method using the compositions and methods disclosed herein.

To begin the procedure, adipose tissue is harvested from the man. Theprocedure is performed at the individual's bedside. The physicianexamines the individual's habitus for a suitable site or sites toharvest adipose tissue and selects the abdomen, and lateral and medialthigh regions. Using a 12-blade scalpel, a small puncture wound is madein order to transverse the dermis. The blade is turned 360 degrees tocomplete the wound. A two-holed blunt harvesting cannula (3 mm innerdiameter) connected to a syringe is then inserted into the subcutaneousadipose tissue plane. The cannula is then moved throughout the plane todisrupt the connective tissue architecture. The volume of aspirateobtained is about 900 mL.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is mixed with the processed adiposetissue. The amount of compound added is an amount sufficient to promoteformation of a blood supply sufficient to support the transplantedtissue. This composition is then transferred to 20 mL syringes.One-holed blunt infiltration cannulas (14-gauge) are used to place about800 mL of adipose tissue transdermally into the bladder neck andproximal urethra regions.

Alternatively, the adipose tissue is first administered into theindividual, and a compound of formula I, such as, e.g., Compound 1,Compound 2, Compound 3, Compound 4, and/or Compound 5, is subsequentlyadministered into the same, or in the vicinity of, the region where theadipose tissue was implanted.

The individual is monitored after the procedure. Approximately threedays after the transplant, the he experiences a decreased frequency ofincontinence. Approximately one month after the procedure, theindividual indicates that his quality of life has improved. Thephysician evaluates the engrafted tissue and determines that thelong-term engraftment was successful.

Example 10 Adipose Tissue Transplant for Breast Defect Correction

This example illustrates the use of compositions and methods disclosedherein for a breast defect correction.

A 56-year-old woman presents with a surgically removed breast due tocancer. Pre-operative evaluation of the person includes routine historyand physical examination in addition to thorough informed consentdisclosing all relevant risks and benefits of the procedure. Thephysician evaluating the individual determines that she is a candidatefor a soft tissue replacement method using the compositions and methodsdisclosed herein.

To begin the procedure, a breast mound is formed using a TRAM-flapprocedure. In this procedure, a portion of abdomen tissue, includingskin, adipose tissue, minor muscles and connective tissues, is takenfrom the patient's abdomen and transplanted onto the breast site. Thistissue is then used to create a breast mound.

A compound of formula I, such as, e.g., Compound 1, Compound 2, Compound3, Compound 4, and/or Compound 5, is then administered into the breastmound region. The amount of compound administered is an amountsufficient to promote formation of a blood supply sufficient to supportthe transplanted tissue.

The individual is monitored for approximately 7 days. The physicianevaluates the engrafted tissue and determines that the engraftment wassuccessful. Both the woman and her physician are satisfied with theresults of the procedure. Approximately one month after the procedure,the woman indicates that her quality of life has improved. Subsequentsurgery is performed to create a nipple and areola.

In closing, it is to be understood that although aspects of the presentspecification have been described with reference to the variousembodiments, one skilled in the art will readily appreciate that thespecific examples disclosed are only illustrative of the principles ofthe subject matter disclosed herein. Therefore, it should be understoodthat the disclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which is defined solely by the claims.Accordingly, the present invention is not limited to that precisely asshown and described.

Certain embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention. Ofcourse, variations on these described embodiments will become apparentto those of ordinary skill in the art upon reading the foregoingdescription. The inventor expects skilled artisans to employ suchvariations as appropriate, and the inventors intend for the invention tobe practiced otherwise than specifically described herein. Accordingly,this invention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

Groupings of alternative elements or embodiments of the inventiondisclosed herein are not to be construed as limitations. Each groupmember may be referred to and claimed individually or in any combinationwith other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, ordeleted from, a group for reasons of convenience and/or patentability.When any such inclusion or deletion occurs, the specification is deemedto contain the group as modified thus fulfilling the written descriptionof all Markush groups used in the appended claims.

Unless otherwise indicated, all numbers expressing quantities ofingredients, properties such as molecular weight, reaction conditions,and so forth used in the specification and claims are to be understoodas being modified in all instances by the term “about.” As used herein,the term “about” means that the item, parameter or term so qualifiedencompasses a range of plus or minus ten percent above and below thevalue of the stated item, parameter or term. Accordingly, unlessindicated to the contrary, the numerical parameters set forth in thespecification and attached claims are approximations that may varydepending upon the desired properties sought to be obtained by thepresent invention. At the very least, and not as an attempt to limit theapplication of the doctrine of equivalents to the scope of the claims,each numerical parameter should at least be construed in light of thenumber of reported significant digits and by applying ordinary roundingtechniques. Notwithstanding that the numerical ranges and parameterssetting forth the broad scope of the invention are approximations, thenumerical values set forth in the specific examples are reported asprecisely as possible. Any numerical value, however, inherently containscertain errors necessarily resulting from the standard deviation foundin their respective testing measurements.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the invention (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention otherwise claimed. No languagein the specification should be construed as indicating any non-claimedelement essential to the practice of the invention.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the invention so claimed areinherently or expressly described and enabled herein.

All patents, patent publications, and other publications referenced andidentified in the present specification are individually and expresslyincorporated herein by reference in their entirety for the purpose ofdescribing and disclosing, for example, the compositions andmethodologies described in such publications that might be used inconnection with the present invention. These publications are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing in this regard should be construed as an admissionthat the inventors are not entitled to antedate such disclosure byvirtue of prior invention or for any other reason. All statements as tothe date or representation as to the contents of these documents isbased on the information available to the applicants and does notconstitute any admission as to the correctness of the dates or contentsof these documents.

The invention claimed is:
 1. A composition comprising: a) adiposetissue; and b) a therapeutically effective amount of a compound of thestructure of formula I

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ alkyl; R⁶is CO₂H, CO₂R⁷, CON(R⁷)₂, CONHCH₂CH₂OH, CON(CH₂CH₂OH)₂, CH₂OR⁷,P(O)(OR⁷)₂,

R⁷ is independently H, C₁-C₆ alkyl or C₂-C₆ alkenyl; X and Y are eachindependently selected from H, OH, ═O, Cl, Br, I, or CF₃; Z¹ and Z² areeach independently selected from CH or N; W¹ and W² are eachindependently selected from CH, CH₂, aryl or substituted aryl,heteroaryl, substituted heteroaryl; m is 0, 1, 2, 3 or 4; o is 0, 1, 2,3, 4, 5 or 6; p is 0 or 1; and V is methyl, aryl, substituted aryl,heteroaryl, or substituted heteroaryl; wherein, when V is methyl, Z¹ orZ² is N; a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable amine salt thereof.
 2. The composition ofclaim 1, wherein the compound has the structure of formula II

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ alkyl; R⁴is H, C₁-C₆ alkyl, C₂-C₆ alkenyl; X and Y are each independentlyselected from H, OH, ═O, Cl, Br, I, or CF₃; Z¹ and Z² are eachindependently selected from CH or N; W¹ and W² are each independentlyselected from CH, CH₂, aryl or substituted aryl, heteroaryl, substitutedheteroaryl; m is 0, 1, 2, 3 or 4; o is 0, 1, 2, 3, 4, 5 or 6; p is 0 or1; and V is methyl, aryl, substituted aryl, heteroaryl, or substitutedheteroaryl; wherein, when V is methyl, Z¹ or Z² is N; a pharmaceuticallyacceptable salt thereof, or a pharmaceutically acceptable amine saltthereof.
 3. The composition of claim 1, wherein V is

wherein U is C, N, O, or S; R⁵ is halogen, C₁-C₆ alkyl, or C₂-C₆alkenyl; and n is 0-7.
 4. The composition of claim 3, wherein U is S; R⁵is F, Cl, Br, or I; and n is 1, 2, or
 3. 5. The composition of claim 1,wherein W² is thiophene.
 6. The composition of claim 1, wherein thecompound has the structure of formula III

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ alkyl; R⁴is H, C₁-C₆ alkyl, C₂-C₆ alkenyl; W¹ and W² are each independentlyselected from CH, CH₂, aryl or substituted aryl, heteroaryl, substitutedheteroaryl; m is 0, 1, 2, 3 or 4; o is 0, 1, 2, 3 or 4; p is 0 or 1; andV is CH₃, aryl, aryl or substituted aryl, heteroaryl, substitutedheteroaryl; a pharmaceutically acceptable salt thereof, apharmaceutically acceptable amine salt thereof.
 7. The composition ofclaim 1, wherein the compound has the structure of formula IV

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ linearalkyl; R⁴ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl; m is 0, 1, 2, 3 or 4; o is0, 1, 2, 3 or 4; p is 0 or 1; and V is CH₃, aryl, aryl or substitutedaryl, heteroaryl, substituted heteroaryl; a pharmaceutically acceptablesalt thereof, or a pharmaceutically acceptable amine salt thereof. 8.The composition of claim 1, wherein the compound has the structure offormula V

wherein each dashed line represents the presence or absence of a bond;R⁴ is H, C₁-C₆ alkyl, C₂-C₆ alkenyl; o is 0, 1, 2, 3 or 4; and V is CH₃,aryl, aryl or substituted aryl, heteroaryl, substituted heteroaryl; apharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable amine salt thereof.
 9. The composition of claim 1, whereinthe compound is

a pharmaceutically acceptable salt thereof, or a pharmaceuticallyacceptable amine salt thereof.
 10. The composition of claim 1, whereinthe compound is incorporated into a drug delivery platform comprising abiodegradable polymer matrix.
 11. The composition of claim 1, whereinthe biodegradable polymer matrix comprises a silk fibroin.
 12. A methodof treating a soft tissue condition of an individual, the methodcomprising the step of administering to a site of the soft tissuecondition a composition comprising adipose tissue and a therapeuticallyeffective amount of a compound of the structure of formula I

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ alkyl; R⁶is CO₂H, CO₂R⁷, CON(R⁷)₂, CONHCH₂CH₂OH, CON(CH₂CH₂OH)₂, CH₂OR⁷,P(O)(OR⁷)₂,

R⁷ is independently H, C₁-C₆ alkyl or C₂-C₆ alkenyl; X and Y are eachindependently selected from H, OH, ═O, Cl, Br, I, or CF₃; Z¹ and Z² areeach independently selected from CH or N; W¹ and W² are eachindependently selected from CH, CH₂, aryl or substituted aryl,heteroaryl, substituted heteroaryl; m is 0, 1, 2, 3 or 4; o is 0, 1, 2,3, 4, 5 or 6; p is 0 or 1; and V is methyl, aryl, substituted aryl,heteroaryl, or substituted heteroaryl; wherein when V is methyl, Z¹ orZ² is N; a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable amine salt thereof, wherein administrationof the composition promotes formation of a blood supply sufficient tosupport the transplanted tissue, thereby treating the soft tissue site.13. The method of claim 12, wherein the soft tissue condition is whereinthe soft tissue condition is a breast tissue condition, a facial tissuecondition, a neck condition, a skin condition, an upper arm condition, alower arm condition, a hand condition, a shoulder condition, a backcondition, a torso including abdominal condition, a buttock condition,an upper leg condition, a lower leg condition including calf condition,a foot condition including plantar fat pad condition, an eye condition,a genital condition, or a condition effecting another body part, regionor area.
 14. The method of claim 13, wherein the breast tissue conditionis a breast imperfection, a breast defect, a breast augmentation, or abreast reconstruction.
 15. The method of claim 13, wherein the facialtissue condition is a facial imperfection, a facial defect, a facialaugmentation, or a facial reconstruction.
 16. The method of claim 13,wherein the facial soft tissue condition is a dermal divot, a sunkencheck, a thin lip, a nasal imperfection or defect, a retro-orbitalimperfection or defect, a facial fold, a facial line, a facial wrinkle,or other size, shape or contour imperfection or defect of the face. 17.The method of claim 12, wherein the soft tissue condition is urinaryincontinence, fecal incontinence, or gastroesophageal reflux disease(GERD).
 18. A method of treating a soft tissue condition of anindividual, the method comprising the steps of a) administering adiposetissue to a site of the soft tissue condition; b) administering atherapeutically effective amount of a compound of the structure offormula I to the site of the soft tissue condition,

wherein each dashed line represents the presence or absence of a bond;R¹, R² and R³ are each independently selected from H or C₁-C₆ alkyl; R⁶is CO₂H, CO₂R⁷, CON(R⁷)₂, CONHCH₂CH₂OH, CON(CH₂CH₂OH)₂, CH₂OR⁷,P(O)(OR⁷)₂,

R⁷ is independently H, C₁-C₆ alkyl or C₂-C₆ alkenyl; X and Y are eachindependently selected from H, OH, ═O, Cl, Br, I, or CF₃; Z¹ and Z² areeach independently selected from CH or N; W¹ and W² are eachindependently selected from CH, CH₂, aryl or substituted aryl,heteroaryl, substituted heteroaryl; m is 0, 1, 2, 3 or 4; o is 0, 1, 2,3, 4, 5 or 6; p is 0 or 1; and V is methyl, aryl, substituted aryl,heteroaryl, or substituted heteroaryl; wherein when V is methyl, Z¹ orZ² is N; a pharmaceutically acceptable salt thereof, or apharmaceutically acceptable amine salt thereof, wherein administrationof the composition promotes formation of a blood supply sufficient tosupport the transplanted tissue, thereby treating the soft tissue site.19. The method of claim 18, wherein the soft tissue condition is whereinthe soft tissue condition is a breast tissue condition, a facial tissuecondition, a neck condition, a skin condition, an upper arm condition, alower arm condition, a hand condition, a shoulder condition, a backcondition, a torso including abdominal condition, a buttock condition,an upper leg condition, a lower leg condition including calf condition,a foot condition including plantar fat pad condition, an eye condition,a genital condition, or a condition effecting another body part, regionor area.
 20. The method of claim 19, wherein the breast tissue conditionis a breast imperfection, a breast defect, a breast augmentation, or abreast reconstruction.
 21. The method of claim 19, wherein the facialtissue condition is a facial imperfection, a facial defect, a facialaugmentation, or a facial reconstruction.
 22. The method of claim 19,wherein the facial soft tissue condition is a dermal divot, a sunkencheck, a thin lip, a nasal imperfection or defect, a retro-orbitalimperfection or defect, a facial fold, a facial line, a facial wrinkle,or other size, shape or contour imperfection or defect of the face. 23.The method of claim 18, wherein the soft tissue condition is urinaryincontinence, fecal incontinence, or gastroesophageal reflux disease(GERD).